Tumor antigens for determining cancer therapy

ABSTRACT

The present invention relates to the treatment of cancer, in particular breast cancer, particularly triple-negative breast cancer. More particularly, the invention concerns methods and means for cancer treatment involving a specific set of tumor antigens.

CROSS REFERENCE TO RELATED APPLICATION

The application is a National Phase Under 35 U.S.C. § 371 ofInternational Application No. PCT/EP2014/066328 filed on Jul. 30, 2014,which claims the benefit of International Application Nos.PCT/EP2013/002271 filed on Jul. 30, 2013 and PCT/EP2013/003173 filedOct. 22, 2013, the entire contents of which are herein incorporated byreference.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Jan. 28, 2016 as a text file named“37592_0002U1_Sequence_Listing” created on Jan. 27, 2016, and having asize of 50,467 bytes is hereby incorporated by reference pursuant to 37C.F.R. § 1.52(e)(5).

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the treatment of cancer, in particularbreast cancer, particularly triple-negative breast cancer. Moreparticularly, the invention concerns methods and means for cancertreatment involving a specific set of tumor antigens.

BACKGROUND OF THE INVENTION

Breast cancer is a type of cancer originating from breast tissue, mostcommonly from the inner lining of milk ducts or the lobules that supplythe ducts with milk. Occasionally, breast cancer presents as metastaticdisease. Common sites of metastasis include bone, liver, lung and brain.Treatment of breast cancer may include surgery, medications (hormonaltherapy and chemotherapy), radiation and/or immunotherapy.

Breast cancer cells may or may not have three important receptors:estrogen receptor (ER), progesterone receptor (PR), and HER2. ERpositive and PR positive breast cancers can be treated with drugs thateither block the receptors, e.g. tamoxifen (Nolvadex), or alternativelyblock the production of estrogen with an aromatase inhibitor, e.g.anastrozole (Arimidex) or letrozole (Femara). Aromatase inhibitors,however, are only suitable for post-menopausal patients. This is becausethe active aromatase in postmenopausal women is different from theprevalent form in premenopausal women, and therefore these agents areineffective in inhibiting the predominant aromatase of premenopausalwomen.

Chemotherapy is predominately used for stage 2-4 disease and isparticularly beneficial in ER negative disease. They are given incombinations, usually for 3-6 months. One of the most common treatmentsis cyclophosphamide plus doxorubicin (adriamycin), known as AC. Mostchemotherapy medications work by destroying fast-growing and/orfast-replicating cancer cells either by causing DNA damage uponreplication or other mechanisms; these drugs also damage fast-growingnormal cells where they cause serious side effects. Damage to the heartmuscle is the most dangerous complication of doxorubicin. Sometimes ataxane drug, such as docetaxel, is added, and the regime is then knownas CAT; taxane attacks the microtubules in cancer cells. Another commontreatment, which produces equivalent results, is cyclophosphamide,methotrexate, and fluorouracil (CMF).

Trastuzumab (Herceptin), a monoclonal antibody to HER2, is onlyeffective in patients with HER2 amplification/overexpression.Trastuzumab, however, is expensive, and approximately 2% of patientssuffer significant heart damage. Other monoclonal antibodies are alsoundergoing clinical trials. Between 25 and thirty percent of breastcancers have an amplification of the HER2 gene or overexpression of itsprotein product. Overexpression of this receptor in breast cancer isassociated with increased disease recurrence and worse prognosis.

Antigen-specific immunotherapy aims to enhance or induce specific immuneresponses in patients and has been successfully used to control cancerdiseases. In particular, T cells play a central role in cell-mediatedimmunity in humans and animals. The recognition and binding of aparticular antigen is mediated by the T cell receptors (TCRs) expressedon the surface of T cells. The T cell receptor (TCR) of a T cell is ableto interact with immunogenic peptides (epitopes) bound to majorhistocompatibility complex (MHC) molecules and presented on the surfaceof target cells. Specific binding of the TCR triggers a signal cascadeinside the T cell leading to proliferation and differentiation into amaturated effector T cell.

The present application relates to the identification of a set of tumorantigens (tumor antigen target portfolio) which is useful in a largefraction of cancer patients, in particular breast cancer patients,particularly triple-negative breast cancer patients. The tumor antigensof said tumor antigen target portfolio are shared tumor antigens whichare expressed in a large fraction of cancer patients. The tumor antigentarget portfolio identified according to the invention may be used fordesigning a drug repository for “off the shelf” pre-manufacturedvaccines (warehouse). These vaccines are useful for treating a largefractions of cancer patients.

Specifically, the present invention may involve the identification ofthe patient-specific expression pattern of the tumor antigen targetportfolio identified according to the invention and selecting a cancertherapy regimen based on said individual expression pattern, preferablyby selecting suitable vaccines from pre-manufactured vaccines targetingexpressed tumor antigens of the tumor antigen target portfolio.Alternatively, the present invention may involve the administration ofpre-manufactured vaccines targeting the tumor antigen target portfolioidentified according to the invention, preferably without prioridentification of the patient-specific expression pattern of this tumorantigen target portfolio.

For vaccination, preferably epitopes from tumor antigens of the tumorantigen target portfolio identified according to the invention areprovided to a patient for presentation by MHC molecules and stimulationof appropriate T cells. In one embodiment, said epitopes are provided asa part of a larger unit such as in the form of an entire tumor antigenor a portion thereof or in the form of a polypeptide comprising saidepitopes and following appropriate processing and presentation by MHCmolecules the epitopes are displayed to the patient's immune system forstimulation of appropriate T cells. The immunogenic products such as thetumor antigens or portions thereof or polypeptides comprising one ormore immunogenic epitopes from one or more tumor antigens of the tumorantigen target portfolio against which an immune response is to beinduced are preferably administered to a patient as RNA encoding theimmunogenic products. In particular, in vitro transcribed RNA (IVT-RNA)may be directly injected into a patient by different immunization routesand following translation of the RNA in transfected cells the expressionproduct following processing may be presented on MHC molecules on thesurface of the cells to elicit an immune response. The advantages ofusing RNA as a kind of reversible gene therapy include transientexpression and a non-transforming character. RNA does not need to enterthe nucleus in order to be expressed and moreover cannot integrate intothe host genome, thereby eliminating the risk of oncogenesis.Transfection rates attainable with RNA are relatively high. Furthermore,the amounts of protein achieved correspond to those in physiologicalexpression.

DESCRIPTION OF INVENTION Summary of the Invention

The present invention relates to methods for treating cancer, inparticular breast cancer, particularly triple-negative breast cancer,involving a specific set of tumor antigens (tumor antigen targetportfolio) expressed in a large fraction of patients. In particular, thepresent invention relates to methods for inducing an immune response ina cancer patient by administering cancer vaccines targeting a tumorantigen target portfolio or tumor antigens selected from a tumor antigentarget portfolio. Preferably, the cancer vaccines administered accordingto the invention to a patient provide MHC presented epitopes which aresuitable for stimulating, priming and/or expanding T cells directedagainst cells expressing antigens of said tumor antigen target portfoliofrom which the MHC presented epitopes are derived. Thus, the vaccinesdescribed herein are preferably capable of inducing or promoting acellular response, preferably cytotoxic T cell activity, against acancer disease characterized by presentation of one or more of saidantigens with class I MHC.

According to the invention, a vaccine for inducing an immune response ispreferably selected from a pre-furnished vaccine warehouse such as apre-furnished RNA vaccine warehouse. This approach is also referred toas “off the shelf” herein. Such pre-furnished vaccine warehouse relatesto a set comprising pre-manufactured vaccine products eachpre-manufactured vaccine product inducing an immune response against atumor antigen recited herein such as CXorf61, CAGE1 or PRAME. Accordingto the invention, such warehouse is designed for being applicable to alarge fraction of cancer patients. Since such vaccine warehouse isselected so as to be applicable to a large fraction of patients, it willbe possible to select from said pre-furnished vaccine warehouse one ormore vaccine products that will induce an immune response against one ormore tumor antigens expressed in cancer cells of a particular patientbeing treated and, thus, targeting the tumor antigen profile of arespective patient without the need of providing further vaccineproducts specifically designed for the patient being treated. Suchselection can be done by testing the patient for tumor antigenexpression and then selecting appropriate vaccine products from thepre-furnished vaccine warehouse targeting said tumor antigens expressedby cancer cells of the patient. Such selection can be made based on thetranscriptomic/peptidomic analysis of the patients tumor. For example,tumor samples from eligible patients can be analysed for their tumorantigen expression patterns. The tumor antigen expression pattern can bedetermined by quantitative, multiplex RT-PCR and IHC and the respectivevaccine products can be selected from the warehouse. Selection from saidpre-furnished vaccine warehouse of one or more vaccine products thatwill induce an immune response against one or more tumor antigensexpressed in cancer cells of a particular patient being treated and,thus, targeting the tumor antigen profile of a respective patient canalso be done by randomly selecting vaccine products from thepre-furnished vaccine warehouse which based on empirical data will mostlikely target one or more tumor antigens expressed by cancer cells ofthe patient. According to the invention the term “tumor antigen profile”refers to a collection of tumor antigens expressed in cancer cells of apatient.

A vaccine for inducing an immune response, when administered to apatent, preferably provides MHC presented epitopes from one or more ofthe tumor antigens mentioned herein. Presentation of these epitopes bycells of a patient, in particular antigen presenting cells, preferablyresults in T cells targeting the epitopes when bound to MHC and thus,the patient's tumor, preferably the primary tumor as well as tumormetastases, expressing antigens from which the MHC presented epitopesare derived and presenting the same epitopes on the surface of the tumorcells.

Administration of a vaccine according to the invention may provide MHCclass II-presented epitopes that are capable of eliciting a CD4+ helperT cell response against cells expressing antigens from which the MHCpresented epitopes are derived. Alternatively or additionally,administration of a vaccine according to the invention may provide MHCclass I-presented epitopes that are capable of eliciting a CD8+ T cellresponse against cells expressing antigens from which the MHC presentedepitopes are derived. Furthermore, administration of a vaccine accordingto the invention may provide one or more neo-epitopes as well as one ormore epitopes not containing cancer specific somatic mutations. In oneembodiment, administration of a vaccine according to the inventionprovides neo-epitopes that are MHC class II-presented epitopes and/orare capable of eliciting a CD4+ helper T cell response against cellsexpressing antigens from which the MHC presented epitopes are derived aswell as epitopes not containing cancer-specific somatic mutations thatare MHC class I-presented epitopes and/or are capable of eliciting aCD8+ T cell response against cells expressing antigens from which theMHC presented epitopes are derived.

According to the invention, epitopes of tumor antigens of the set oftumor antigens identified according to the invention may be present in avaccine as polyepitopic polypeptide or nucleic acid such as RNA encodingsuch polyepitopic polypeptide. If it is intended to use apre-manufactured polyepitopic polypeptide for inducing an immuneresponse, the polyepitopic polypeptide is preferably administeredwithout determining the patient's individual tumor antigen expressionpattern. The polyepitopic polypeptide comprises epitopes which based onempirical data will most likely induce an immune response targeting oneor more tumor antigens expressed by cancer cells of the patient. To thisend, it is demonstrated in the examples that a specific set of onlythree different tumor antigens is sufficient so as to cover 95% of thetriple-negative breast cancer (TNBC) patient samples analyzed. In otherwords, 95% of the TNBC patients express at least one antigen which isamong said specific set of only three different tumor antigens. Thus,including into a polyepitopic polypeptide at least one epitope from eachof said three different tumor antigens would be expected to induce animmune response in 95% of the TNBC patients.

One aspect of the invention relates to a method for treating cancer in apatient comprising determining the expression pattern of a set of tumorantigens and selecting a cancer therapy regimen based on the expressionpattern determined, wherein the set of tumor antigens comprises CXorf61and CAGE1. In one embodiment, the set of tumor antigens furthercomprises PRAME. In another embodiment, the set of tumor antigensfurther comprises one or more antigens selected from the groupconsisting of CBX2, PLAC1, CLDN6, SPANX, MAGEA3, TPTE, ACTL8, ANKRD30A,CDKN2A, MAD2L1, CTAG1B, MAGEA4, MAGEA5, SUNC1, MAGEA10, LRRN1 andMAGEA9.

In one embodiment, the expression pattern is determined in a sampleobtained from a cancer patient. In one embodiment, the sample comprisescancer cells. In one embodiment, the sample is a tumor specimen of thecancer patient.

In one embodiment, determining the expression pattern comprises aquantitative and/or qualitative determination of the expression of thetumor antigens. In one embodiment, determining the expression patterncomprises determining the expression of RNA and/or protein of the tumorantigens.

In one embodiment, the cancer therapy regimen based on the expressionpattern determined comprises immunotherapeutically targeting those tumorantigens of the set of tumor antigens which are expressed in cancercells of the patient. In one embodiment, the cancer therapy regimenbased on the expression pattern determined comprises inducing an immuneresponse in the patient against those tumor antigens of the set of tumorantigens which are expressed in cancer cells of the patient. In oneembodiment, the immune response is induced by providing to the patientone or more immunogenic epitopes of each of those tumor antigens of theset of tumor antigens which are expressed in cancer cells of thepatient.

In one embodiment, the immune response comprises a cellular response. Inone embodiment, the immune response comprises a CD8+ T cell responseand/or a CD4+ T cell response. In one embodiment, the cellular responseis induced by administering to the patient a vaccine providing one ormore T cell epitopes of each of those tumor antigens of the set of tumorantigens which are expressed in cancer cells of the patient. In oneembodiment, the one or more T cell epitopes are comprised in the vaccinein one or more peptides or polypeptides wherein said one or morepeptides or polypeptides following administration are processed toproduce the one or more T cell epitopes. In one embodiment, the vaccineis an RNA vaccine. In one embodiment, following appropriate processingand presentation by MHC molecules the T cell epitopes are displayed tothe patient's immune system for stimulation of appropriate T cells.

A further aspect of the invention relates to a method for preventing ortreating cancer in a patient comprising immunotherapeutically targetingeach tumor antigen of a set of tumor antigens, wherein the set of tumorantigens comprises CXorf61 and CAGE1. In one embodiment, the set oftumor antigens further comprises PRAME. In another embodiment, the setof tumor antigens further comprises one or more antigens selected fromthe group consisting of CBX2, PLAC1, CLDN6, SPANX, MAGEA3, TPTE, ACTL8,ANKRD30A, CDKN2A, MAD2L1, CTAG1B, MAGEA4, MAGEA5, SUNC1, MAGEA10, LRRN1and MAGEA9.

In one embodiment, the method of the invention comprises inducing animmune response in the patient against each tumor antigen of the set oftumor antigens. In one embodiment, the immune response is induced byproviding to the patient one or more immunogenic epitopes of each tumorantigen of the set of tumor antigens.

In one embodiment, the immune response comprises a cellular response. Inone embodiment, the immune response comprises a CD8+ T cell responseand/or a CD4+ T cell response. In one embodiment, the cellular responseis induced by administering a vaccine providing one or more T cellepitopes of each tumor antigen of the set of tumor antigens to thepatient. In one embodiment, the one or more T cell epitopes arecomprised in the vaccine in one or more peptides or polypeptides whereinsaid one or more peptides or polypeptides following administration areprocessed to produce the one or more T cell epitopes. In one embodiment,the vaccine is an RNA vaccine. In one embodiment, following appropriateprocessing and presentation by MHC molecules the T cell epitopes aredisplayed to the patient's immune system for stimulation of appropriateT cells.

In one embodiment of all aspects of the invention, the cancer is breastcancer. In one embodiment of all aspects of the invention, the cancer istriple-negative breast cancer.

A further aspect of the invention relates to a set of vaccine productscomprising vaccine products for inducing an immune response against eachtumor antigen of a set of tumor antigens in a patient, wherein the setof tumor antigens comprises CXorf61 and CAGE1. In one embodiment, theset of tumor antigens further comprises PRAME. In another embodiment,the set of tumor antigens further comprises one or more antigensselected from the group consisting of CBX2, PLAC1, CLDN6, SPANX, MAGEA3,TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, MAGEA4, MAGEA5, SUNC1,MAGEA10, LRRN1 and MAGEA9.

In one embodiment, the vaccine products when administered to a patientprovide one or more immunogenic epitopes of each tumor antigen of theset of tumor antigens.

In one embodiment, the immune response comprises a cellular response. Inone embodiment, the immune response comprises a CD8+ T cell responseand/or a CD4+ T cell response. In one embodiment, the vaccine productswhen administered to a patient provide one or more T cell epitopes ofeach tumor antigen of the set of tumor antigens. In one embodiment, theset of vaccine products of the invention comprises one or more peptidesor polypeptides comprising the one or more T cell epitopes. In oneembodiment, following appropriate processing and presentation by MHCmolecules the T cell epitopes are displayed to the patients immunesystem for stimulation of appropriate T cells. In one embodiment, thevaccine products are RNA vaccines.

A further aspect of the invention relates to a kit for determining theexpression pattern of a set of tumor antigens, wherein the set of tumorantigens comprises CXorf61 and CAGE1 and wherein the kit comprisesreagents specifically binding to each tumor antigen of said set of tumorantigens or nucleic acids coding therefor. In one embodiment, the set oftumor antigens further comprises PRAME and the set of reagents furthercomprises reagents specifically binding to PRAME or a nucleic acidcoding therefor. In another embodiment, the set of tumor antigensfurther comprises one or more antigens selected from the groupconsisting of CBX2, PLAC1, CLDN6, SPANX, MAGEA3, TPTE, ACTL8, ANKRD30A,CDKN2A, MAD2L1, CTAG1B, MAGEA4, MAGEA5, SUNC1, MAGEA10, LRRN1 and MAGEA9and the set of reagents further comprises reagents specifically bindingto said tumor antigens or nucleic acid coding therefor.

In one embodiment, the immunogenic epitopes used according to theinvention may have patient-specific amino acid sequence modificationswhich may be based on cancer specific somatic mutations present incancer cells of the patient. Thus, the present invention may involve:(a) identifying cancer specific somatic mutations in a tumor specimen ofa cancer patient; and (b) using for immunization immunogenic epitopes ornucleic acids encoding said epitopes incorporating sequencemodifications resulting from cancer specific somatic mutationsdetermined in step (a).

Thus, a vaccine for inducing an immune response, when administered to apatent, may provide epitopes incorporating sequence changes based on theidentified mutations or sequence differences. Such MHC presentedepitopes incorporating sequence changes based on the identifiedmutations or sequence differences are also termed “neo-epitopes” herein.

The vaccines described herein may comprise a pharmaceutically acceptablecarrier and may optionally comprise one or more adjuvants, stabilizersetc. The vaccines may in the form of a therapeutic or prophylacticvaccine.

In further aspects, the invention provides the vaccines described hereinfor use in the methods of treatment described herein, in particular foruse in treating or preventing cancer.

The treatments of cancer described herein can be combined with surgicalresection and/or radiation and/or traditional chemotherapy.

A further aspect of the invention relates to a method for determiningthe expression of the tumor antigen CXorf61 comprising the step ofassaying a sample with respect to expression of a nucleic acid whichcomprises the nucleic acid sequence of SEQ ID NO: 29 or a variant ofsaid nucleic acid sequence. Accordingly, if according to the inventionthe expression (pattern) of CXorf61 or a set of tumor antigenscomprising CXorf61 is to be determined, said determination may comprisethe step of assaying a sample with respect to expression of a nucleicacid which comprises the nucleic acid sequence of SEQ ID NO: 29 or avariant of the nucleic acid sequence.

A further aspect of the invention relates to a method for diagnosis,detection or monitoring, i.e. determining the regression, progression,course and/or onset, of cancer comprising the step of assaying a samplewith respect to expression of a nucleic acid which comprises the nucleicacid sequence of SEQ ID NO: 29 or a variant of said nucleic acidsequence.

In the above aspects, assaying a sample with respect to expression of anucleic acid may comprise a quantitative and/or qualitativedetermination of the expression of the nucleic acid.

Accordingly, the present invention relates to a method for diagnosis,detection or monitoring of cancer comprising the detection of and/ordetermination of the quantity of a nucleic acid which comprises thenucleic acid sequence of SEQ ID NO: 29 or a variant of said nucleic acidsequence, in a sample isolated from a patient, preferably from a patienthaving cancer, being suspected of having or falling ill with cancer orhaving a potential for cancer.

Preferably, the above methods involve the use of ligands such as nucleicacid probes which specifically bind to the nucleic acid which is to bedetected or the quantity of which is to be determined. The above methodsmay be used to detect whether a subject has or is at (increased) risk ofdeveloping cancer, or, for instance, whether a treatment regimen isefficient.

Preferably, the cancer which is to be diagnosed, detected or monitoredusing the methods of the invention is selected from the group consistingof ovarian cancer, lung cancer, gastric cancer, breast cancer, hepaticcancer, pancreatic cancer, esophageal cancer and prostate cancer. In aparticularly preferred embodiment, the cancer is breast cancer, inparticular triple-negative breast cancer.

Preferably, the cancer which is to be diagnosed, detected or monitoredusing the methods of the invention is a cancer disease wherein cancercells express the tumor antigen CXorf61, more preferably wherein cancercells express or aberrantly express the tumor antigen CXorf61 whilecells of the non-cancerous tissue preferably do not express the tumorantigen CXorf61 or express the tumor antigen CXorf61 at a lower level.

Typically, the level of the target nucleic acid (i.e. the nucleic acidwhich is to be assayed or detected or the quantity of which is to bedetermined in the methods of the invention) in a sample is compared to areference level. Preferably, a deviation from said reference level isindicative of the presence and/or stage of cancer in a subject. Thereference level may be a level determined in one or more referencesamples (e.g., samples from healthy tissues or subjects) or a medianlevel from healthy tissues or subjects. Preferably, a test sample and(a) reference sample(s) are derived from the same tissue type and/or thereference level is a level determined in the same tissue type as thetissue to be tested (from which the test sample is derived). Preferably,a test sample is derived from a tissue which is to be diagnosed,detected or monitored with respect to cancer; e.g. the cancer which isto be diagnosed, detected or monitored is breast cancer and the sampleand optionally reference sample(s) are derived from breast tissue.Preferably, the presence of the target nucleic acid in the sample or aquantity of the target nucleic acid in the sample which is increasedcompared to a reference level, e.g. compared to a patient withoutcancer, indicates the presence of or risk for (i.e. a potential for adevelopment of) cancer.

In one embodiment, a sample in the above methods comprises cancer cells.In one embodiment, the sample is a tumor specimen of a cancer patient.

In one embodiment of the methods of the invention the sample is from atissue or organ wherein the cells when the tissue or organ is free ofcancer do not substantially express CXorf61.

In one embodiment, the sample is from a tissue or organ wherein thecells when the tissue or organ is free of cancer do not substantiallyexpress CXorf61 and the tissue or organ optionally has already beendiagnosed as being affected by cancer, e.g. by visual inspection orculture testing of cells of said tissue or organ. In this embodiment,the presence of the target nucleic acid and/or a quantity of the targetnucleic acid which is increased compared to a reference level, e.g.compared to a patient without cancer, may indicate that the cancer canbe treated by immunotherapeutically targeting CXorf61 using, forexample, the methods and means described herein.

The above methods preferably allow quantitative and/or qualitativeevaluations, e.g., absolute and/or relative measurements of targetnucleic acid.

If reference is made herein to the detection of or the determination ofthe quantity of a nucleic acid, the nucleic acid which is actually to bedetected or the quantity of which is actually to be determined ispreferably mRNA. However, it should be understood that this may alsoinclude embodiments wherein mRNA is detected or the quantity of mRNA isdetermined indirectly. For example, mRNA may be transformed into cDNAand the cDNA is detected or its quantity determined. mRNA is givenherein as the cDNA equivalent. One skilled in the art would understandthat the cDNA sequence is equivalent to the mRNA sequence, and can beused for the same purpose herein, e.g., the generation of probeshybridizing to the nucleic acid to be detected. Thus, if reference ismade herein to the sequences shown in the sequence listing this is alsoto include the RNA equivalents of said sequences.

Means for detection and/or determination of the quantity of targetnucleic acid are described herein and will be apparent to the skilledperson.

Preferably, the detection and/or determination of the quantity of targetnucleic acid comprises (i) contacting a sample with an agent which bindsspecifically to the target nucleic acid, and (ii) detecting theformation of and/or determining the quantity of a complex between theagent and the target nucleic acid.

Typically, the detection and/or determination of the quantity of thetarget nucleic acid involves the use of labeled ligands whichspecifically bind to the target nucleic acid, e.g. a labeled nucleicacid probe that hybridizes to the target nucleic acid.

According to the invention, detection of a nucleic acid or determiningthe quantity of a nucleic acid may be carried out using known nucleicacid detection methods such as methods involving hybridization ornucleic acid amplification techniques. In one embodiment, mRNAtranscripts are detected or the quantity thereof is determined usingRT-PCR or Northern blot analysis.

Such nucleic acid detection methods may involve the use ofoligonucleotides hybridizing to the target nucleic acid. Suitableoligonucleotides typically vary in length from five to several hundrednucleotides, more typically about 20-70 nucleotides in length orshorter, even more typically about 10-30 nucleotides in length.

The methods of monitoring according to the invention preferably comprisea detection of and/or determination of the quantity of the targetnucleic acid in a first sample at a first point in time and in a furthersample at a second point in time, wherein a regression, progression,course and/or onset of cancer may be determined by comparing the twosamples.

A quantity of the target nucleic acid which is decreased in a samplecompared to a sample taken earlier from a patient may indicate aregression, a positive course, e.g. a successful treatment, or a reducedrisk for an onset of cancer in said patient.

A quantity of the target nucleic acid which is increased in a samplecompared to a sample taken earlier from a patient may indicate aprogression, a negative course, e.g. an unsuccessful treatment,recurrence or metastatic behavior, an onset or a risk for an onset ofcancer in said patient.

A further aspect of the invention relates to a kit useful in the abovemethods. These kits in one embodiment comprise a ligand thatspecifically binds to the target nucleic acid; i.e. a nucleic acid whichcomprises the nucleic acid sequence of SEQ ID NO: 29 or a variant ofsaid nucleic acid sequence. In a particular embodiment, the ligandcomprises nucleic acid primers or probes specific for the target nucleicacid as described above. Kits may include informative pamphlets, forexample, pamphlets informing how to use reagents to practice a methoddisclosed herein.

In a further aspect, the invention relates to a nucleic acid moleculesuch as a recombinant nucleic acid molecule, in particular DNA or RNAmolecule, which comprises the nucleic acid sequence of SEQ ID NO: 29 ora variant of said nucleic acid sequence.

The invention also relates to host cells which comprise the nucleic acidmolecule of the invention. Preferably, such host cells express theprotein encoded by the nucleic acid molecule. In one embodiment, thehost cell is an antigen-presenting cell, in particular a dendritic cell,a monocyte or a macrophage.

The above aspects and disclosure with respect to CXorf61 and the nucleicacid sequence of SEQ ID NO: 29 analogously also apply to CAGE1 whereinthe nucleic acid sequence of SEQ ID NOs: 41 or 42 or a variant of saidnucleic acid sequences are concerned. For example, a further aspect ofthe invention relates to a method for determining the expression of thetumor antigen CAGE1 comprising the step of assaying a sample withrespect to expression of a nucleic acid which comprises the nucleic acidsequence of SEQ ID NO: 41 or 42 or a variant of said nucleic acidsequences.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,(1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of biochemistry, cell biology,immunology, and recombinant DNA techniques which are explained in theliterature in the field (cf, e.g., Molecular Cloning: A LaboratoryManual, 2^(nd) Edition, J. Sambrook et al. eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps although in some embodiments suchother member, integer or step or group of members, integers or steps maybe excluded, i.e. the subject-matter consists in the inclusion of astated member, integer or step or group of members, integers or steps.The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”), provided herein is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed. No language in the specification should be construedas indicating any non-claimed element essential to the practice of theinvention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

According to the present invention, the term “peptide” refers tosubstances comprising two or more, preferably 3 or more, preferably 4 ormore, preferably 6 or more, preferably 8 or more, preferably 10 or more,preferably 13 or more, preferably 16 more, preferably 21 or more and upto preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acidsjoined covalently by peptide bonds. The term “polypeptide” or “protein”refers to large peptides, preferably to peptides with more than 100amino acid residues, but in general the terms “peptide”, “polypeptide”and “protein” are synonyms and are used interchangeably herein.

According to the invention, the term “modification” with respect topeptides, polypeptides or proteins relates to a sequence change in apeptide, polypeptide or protein compared to a parental sequence such asthe sequence of a wildtype peptide, polypeptide or protein. The termincludes amino acid insertion variants, amino acid addition variants,amino acid deletion variants and amino acid substitution variants,preferably amino acid substitution variants. All these sequence changesmay potentially create new epitopes.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence.

Amino acid addition variants comprise amino- and/or carboxy-terminalfusions of one or more amino acids, such as 1, 2, 3, 4 or 5, or moreamino acids.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence, such as by removal of 1, 2, 3, 4 or5, or more amino acids.

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place.

The term “derived” means according to the invention that a particularentity, in particular a particular peptide sequence, is present in theobject from which it is derived. In the case of amino acid sequences,especially particular sequence regions, “derived” in particular meansthat the relevant amino acid sequence is derived from an amino acidsequence in which it is present.

The term “immune response” refers to an integrated bodily response to atarget such as an antigen or a cell expressing an antigen and preferablyrefers to a cellular immune response or a cellular as well as a humoralimmune response. The immune response may beprotective/preventive/prophylactic and/or therapeutic.

“Inducing an immune response” may mean that there was no immune responsebefore induction, but it may also mean that there was a certain level ofimmune response before induction and after induction said immuneresponse is enhanced. Thus, “inducing an immune response” also includes“enhancing an immune response”. Preferably, after inducing an immuneresponse in a subject, said subject is protected from developing adisease such as a cancer disease or the disease condition is amelioratedby inducing an immune response. For example, an immune response againsta tumor antigen may be induced in a patient having a cancer disease orin a subject being at risk of developing a cancer disease. Inducing animmune response in this case may mean that the disease condition of thesubject is ameliorated, that the subject does not develop metastases, orthat the subject being at risk of developing a cancer disease does notdevelop a cancer disease.

According to the invention, “inducing an immune response against” inconnection with particular tumor antigens such as CXorf61, CAGE1 and/orPRAME preferably relates to the ability of inducing an immune response,preferably a T cell response against said tumor antigens or cells suchas cancer cells expressing and/or presenting said tumor antigens in apatient. Thus, a vaccine for inducing an immune response against a tumorantigen may comprise a peptide or polypeptide comprising one or moreimmunogenic epitopes of the tumor antigen such as a polypeptidecomprising the tumor antigen or a portion thereof, in particular aportion comprising an immunogenic epitope of the tumor antigen. In oneparticularly preferred embodiment, such peptide or polypeptide accordingto the present invention is administered to a patient in the form of anucleic acid, preferably RNA such as in vitro transcribed or syntheticRNA, which may be expressed in cells of a patient such as antigenpresenting cells to produce the peptide or polypeptide.

The terms “cellular immune response” and “cellular response” or similarterms refer to an immune response directed to cells characterized bypresentation of an antigen with class I or class II MHC involving Tcells or T-lymphocytes which act as either “helpers” or “killers”. Thehelper T cells (also termed CD4⁺ T cells) play a central role byregulating the immune response and the killer cells (also termedcytotoxic T cells, cytolytic T cells, CD8⁺ T cells or CTLs) killdiseased cells such as cancer cells, preventing the production of morediseased cells. In preferred embodiments, the present invention involvesthe stimulation of an anti-tumor CTL response against tumor cellsexpressing one or more tumor antigens and preferably presenting suchtumor antigens with class I MHC.

An “antigen” according to the invention covers any substance, preferablya peptide or protein, that is a target of and/or induces an immuneresponse such as a specific reaction with antibodies or T-lymphocytes (Tcells). Preferably, an antigen comprises at least one epitope such as aT cell epitope. Preferably, an antigen in the context of the presentinvention is a molecule which, optionally after processing, induces animmune reaction, which is preferably specific for the antigen (includingcells expressing the antigen). The antigen or a T cell epitope thereofis preferably presented by a cell, preferably by an antigen presentingcell which includes a diseased cell, in particular a cancer cell, in thecontext of MHC molecules, which results in an immune response againstthe antigen (including cells expressing the antigen).

In one embodiment, an antigen is a tumor antigen (also termedtumor-expressed antigen herein), i.e., a part of a tumor cell such as aprotein or peptide expressed in a tumor cell which may be derived fromthe cytoplasm, the cell surface or the cell nucleus, in particular thosewhich primarily occur intracellularly or as surface antigens of tumorcells. For example, tumor antigens include CXorf61, CAGE1 and PRAME.According to the present invention, a tumor antigen preferably comprisesany antigen which is expressed in and optionally characteristic withrespect to type and/or expression level for tumors or cancers as well asfor tumor or cancer cells, i.e. a tumor-associated antigen. In oneembodiment, the term “tumor-associated antigen” relates to proteins thatare under normal conditions specifically expressed in a limited numberof tissues and/or organs or in specific developmental stages, forexample, the tumor-associated antigens may be under normal conditionsspecifically expressed in stomach tissue, preferably in the gastricmucosa, in reproductive organs, e.g., in testis, in trophoblastictissue, e.g., in placenta, or in germ line cells, and are expressed oraberrantly expressed in one or more tumor or cancer tissues. In thiscontext, “a limited number” preferably means not more than 3, morepreferably not more than 2. The tumor antigens in the context of thepresent invention include, for example, differentiation antigens,preferably cell type specific differentiation antigens, i.e., proteinsthat are under normal conditions specifically expressed in a certaincell type at a certain differentiation stage, cancer/testis antigens,i.e., proteins that are under normal conditions specifically expressedin testis and sometimes in placenta, and germ line specific antigens.Preferably, the tumor antigen or the aberrant expression of the tumorantigen identifies cancer cells. In the context of the presentinvention, the tumor antigen that is expressed by a cancer cell in asubject, e.g., a patient suffering from a cancer disease, is preferablya self-protein in said subject. In preferred embodiments, the tumorantigen in the context of the present invention is expressed undernormal conditions specifically in a tissue or organ that isnon-essential, i.e., tissues or organs which when damaged by the immunesystem do not lead to death of the subject, or in organs or structuresof the body which are not or only hardly accessible by the immunesystem.

According to the invention, the terms “tumor antigen”, “tumor-expressedantigen”, “cancer antigen” and “cancer-expressed antigen” areequivalents and are used interchangeably herein.

The term “CXorf61” relates to a protein comprising, preferablyconsisting of the amino acid sequence of SEQ ID NO: 3 of the sequencelisting or a variant of said amino acid sequence and to a gene thatencodes the protein. According to the invention, peptides comprising,preferably consisting of the amino acid sequence of SEQ ID NO: 10 or 11of the sequence listing or variants of said amino acid sequences areuseful as T cell epitopes for eliciting an immune response against cellsexpressing CXorf61.

The term “CAGE1” relates to a protein comprising, preferably consistingof the amino acid sequence of SEQ ID NO: 4, 5, 6, 7, 8, 43 or 44,preferably SEQ ID NO: 4, 5, 6, 7 or 8 of the sequence listing or avariant of said amino acid sequence and to a gene that encodes theprotein.

The term “PRAME” relates to a protein comprising, preferably consistingof the amino acid sequence of SEQ ID NO: 9 of the sequence listing or avariant of said amino acid sequence and to a gene that encodes theprotein. According to the invention, peptides comprising, preferablyconsisting of the amino acid sequence of SEQ ID NO: 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23 or 24 of the sequence listing or variants ofsaid amino acid sequences are useful as T cell epitopes for eliciting animmune response against cells expressing PRAME.

According to the invention, the term “neoantigen” relates to an antigenincluding one or more amino acid modifications compared to the parentalantigen. For example, a neoantigen may be a tumor-associated neoantigen,wherein the term “tumor-associated neoantigen” includes a peptide orprotein including amino acid modifications due to tumor-specificmutations.

According to the invention, the term “tumor-specific mutation” or“cancer-specific mutation” relates to a somatic mutation that is presentin the nucleic acid of a tumor or cancer cell but absent in the nucleicacid of a corresponding normal, i.e. non-tumorous or non-cancerous,cell. The terms “tumor-specific mutation” and “tumor mutation” and theterms “cancer-specific mutation” and “cancer mutation” are usedinterchangeably herein.

According to the invention, the term “tumor antigen-positive cancer” or“tumor antigen-positive tumor” or similar terms means a cancer or tumorinvolving cancer or tumor cells expressing a tumor antigen.

The term “immunogenicity” relates to the relative effectively to inducean immune response that is preferably associated with therapeutictreatments, such as treatments against cancers. As used herein, the term“immunogenic” relates to the property of having immunogenicity. Forexample, the term “immunogenic” when used in the context of a peptide,polypeptide or protein relates to the effectively of said peptide,polypeptide or protein to induce an immune response that is caused byand/or directed against said peptide, polypeptide or protein.

According to the invention, the immunotherapeutic targeting of anantigen such as a tumor antigen can be effected by any means whichresult in an immune response or immune reaction targeting said antigen,including cells expressing said antigen and optionally presenting theantigen in the context of MHC molecules. Such immunotherapeutictargeting provides for the selective eradication of cells that expressthe antigen and optionally present said antigen, thereby minimizingadverse effects to normal cells not expressing and optionally notpresenting said antigen.

According to the invention, the term “immunotherapeutic targeting”, inparticular, relates to any therapy involving the immune system,components thereof or immune mechanisms that can be used to targetpreferentially diseased cells such as cells expressing tumor antigen(s)and optionally presenting tumor antigen(s) such as cancer cells whilenon-diseased cells not expressing tumor antigen(s) are not targeted ortargeted to a lesser extent. Targeting of diseased cells preferablyresults in killing and/or impairment of proliferation or viability ofdiseased cells.

According to the invention active and/or passive immunotherapeuticstrategies are envisioned for immunotherapeutic targeting. Activeimmunotherapeutic strategies may aim to induce, i.e. activate orsensitize, and expand antigen-specific T cells in the patient, which areable to specifically recognize and kill diseased cells. Differentantigen formats can be used for tumor vaccination including proteins,peptides or nucleic acids such as RNA that can be applied eitherdirectly in vivo or in vitro by pulsing of DCs following transfer intothe patient. Passive immunotherapeutic strategies rely, for example, onthe adoptive transfer of immunoreactive cells, such as lymphoid cells,in particular T cells. T cells may optionally be engineered in vitro toexpress a defined antigen-specific T cell receptor (TCR) specificallytargeting a tumor antigen (presented in the context of MHC molecules).Nucleic acids such as RNA encoding T cell receptor (TCR) chains may beintroduced into T cells. In a suitable embodiment, the TCR α- andβ-chains are cloned out from an antigen-specific T cell line and usedfor adoptive T cell therapy. The T cell receptors are preferablyspecific for an antigen which includes specificity for a peptidefragment derived from the antigen and presented in the context of MHCmolecules. In general, the T cell receptors recognize or bind antigenpeptides presented in the context of MHC. The nucleic acids encoding α-and β-chains of a T cell receptor may be contained on separate nucleicacid molecules such as expression vectors or alternatively, on a singlenucleic acid molecule. Accordingly, the term “a nucleic acid encoding aT cell receptor” relates to nucleic acid molecules encoding the T cellreceptor chains on the same or preferably on different nucleic acidmolecules. According to the invention, T cells may be stimulated, primedand/or expanded in vitro or in vivo. The T cells used for treatmentaccording to the invention may be autologous, allogeneic or syngeneic toa treated subject.

Adoptive cell transfer (ACT) based immunotherapy can be broadly definedas a form of passive immunization with previously sensitized cells, inparticular T cells, that are transferred to recipients or to theautologous host after ex vivo expansion from low precursor frequenciesto clinically relevant cell numbers. Since the antigenic specificity ofT cells is rested entirely on the heterodimeric complex of the TCR α-and β-chain, the transfer of cloned TCR genes into T cells offers thepotential to redirect them towards any antigen of interest. Therefore,TCR gene therapy provides an attractive strategy to developantigen-specific immunotherapy with autologous lymphocytes as treatmentoption.

Other forms of immunotherapeutic strategies may involve targeting ofdisease-associated antigen(s) such as cell surface antigen(s) ondiseased cells by antibodies, antibody fragments, or antibodyderivatives that are either naked or conjugated to a therapeutic moietysuch as cytotoxins or radionuclides. Antibodies, antibody fragments, orantibody derivatives that are not conjugated to a therapeutic moiety mayact through recruiting the patient's immune system to destroy tumorcells, e.g. by inducing complement dependent cytotoxicity (CDC) and/orantibody-dependent cell-mediated cytotoxicity (ADCC).

Strategies involving antibodies may be applied as active and/or passiveimmunotherapeutic strategies.

The terms “major histocompatibility complex” and the abbreviation “MHC”include MHC class I and MHC class II molecules and relate to a complexof genes which occurs in all vertebrates. MHC proteins or molecules areimportant for signaling between lymphocytes and antigen presenting cellsor diseased cells in immune reactions, wherein the MHC proteins ormolecules bind peptides and present them for recognition by T cellreceptors. The proteins encoded by the MHC are expressed on the surfaceof cells, and display both self antigens (peptide fragments from thecell itself) and non-self antigens (e.g., fragments of invadingmicroorganisms) to a T cell.

The MHC region is divided into three subgroups, class I, class II, andclass III. MHC class I proteins contain an α-chain and β2-microglobulin(not part of the MHC encoded by chromosome 15). They present antigenfragments to cytotoxic T cells. On most immune system cells,specifically on antigen-presenting cells, MHC class II proteins containα- and β-chains and they present antigen fragments to T-helper cells,MHC class III region encodes for other immune components, such ascomplement components and some that encode cytokines. The MHC is bothpolygenic (there are several MHC class I and MHC class II genes) andpolymorphic (there are multiple alleles of each gene).

As used herein, the term “haplotype” refers to the HLA alleles found onone chromosome and the proteins encoded thereby. Haplotype may alsorefer to the allele present at any one locus within the MHC. Each classof MHC is represented by several loci: e.g., HLA-A (Human LeukocyteAntigen-A), HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K,HLA-L, HLA-P and HLA-V for class I and HLA-DRA, HLA-DRB1-9, HLA-,HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DMA, HLA-DMB, HLA-DOA, andHLA-DOB for class II. The terms “HLA allele” and “MHC allele” are usedinterchangeably herein.

The MHCs exhibit extreme polymorphism: within the human population thereare, at each genetic locus, a great number of haplotypes comprisingdistinct alleles. Different polymorphic MHC alleles, of both class I andclass II, have different peptide specificities: each allele encodesproteins that bind peptides exhibiting particular sequence patterns.

In one preferred embodiment of all aspects of the invention an MHCmolecule is an HLA molecule.

In the context of the present invention, the term “MHC binding peptide”includes MHC class I and/or class II binding peptides or peptides thatcan be processed to produce MHC class I and/or class II bindingpeptides. In the case of class I MHC/peptide complexes, the bindingpeptides are typically 8-12, preferably 8-10 amino acids long althoughlonger or shorter peptides may be effective. In the case of class IIMHC/peptide complexes, the binding peptides are typically 9-30,preferably 10-25 amino acids long and are in particular 13-18 aminoacids long, whereas longer and shorter peptides may be effective.

If a peptide is to be presented directly, i.e., without processing, inparticular without cleavage, it has a length which is suitable forbinding to an MHC molecule, in particular a class I MHC molecule, andpreferably is 7-30 amino acids in length such as 7-20 amino acids inlength, more preferably 7-12 amino acids in length, more preferably 8-11amino acids in length, in particular 9 or 10 amino acids in length.

If a peptide is part of a larger entity comprising additional sequences,e.g. of a vaccine sequence or polypeptide, and is to be presentedfollowing processing, in particular following cleavage, the peptideproduced by processing has a length which is suitable for binding to anMHC molecule, in particular a class I MHC molecule, and preferably is7-30 amino acids in length such as 7-20 amino acids in length, morepreferably 7-12 amino acids in length, more preferably 8-11 amino acidsin length, in particular 9 or 10 amino acids in length. Preferably, thesequence of the peptide which is to be presented following processing isderived from the amino acid sequence of an antigen or polypeptide usedfor vaccination, i.e., its sequence substantially corresponds and ispreferably completely identical to a fragment of the antigen orpolypeptide.

Thus, an MHC binding peptide in one embodiment comprises a sequencewhich substantially corresponds and is preferably completely identicalto a fragment of an antigen.

The term “epitope” refers to an antigenic determinant in a molecule suchas an antigen, i.e., to a part in or fragment of the molecule that isrecognized by the immune system, for example, that is recognized by a Tcell, in particular when presented in the context of MHC molecules. Anepitope of a protein such as a tumor antigen preferably comprises acontinuous or discontinuous portion of said protein and is preferablybetween 5 and 100, preferably between 5 and 50, more preferably between8 and 30, most preferably between 10 and 25 amino acids in length, forexample, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It isparticularly preferred that the epitope in the context of the presentinvention is a T cell epitope.

According to the invention an epitope may bind to MHC molecules such asMHC molecules on the surface of a cell and thus, may be a “MHC bindingpeptide”.

As used herein the term “neo-epitope” refers to an epitope that is notpresent in a reference such as a normal non-cancerous or germline cellbut is found in cancer cells. This includes, in particular, situationswherein in a normal non-cancerous or germline cell a correspondingepitope is found, however, due to one or more mutations in a cancer cellthe sequence of the epitope is changed so as to result in theneo-epitope.

As used herein, the term “T cell epitope” refers to a peptide whichbinds to a MHC molecule in a configuration recognized by a T cellreceptor. Typically, T cell epitopes are presented on the surface of anantigen-presenting cell.

According to the invention, a T cell epitope may be present in a vaccineas a part of a larger entity such as a vaccine sequence and/or apolypeptide comprising more than one T cell epitope. The presentedpeptide or T cell epitope is produced following suitable processing.

T cell epitopes may be modified at one or more residues that are notessential for TCR recognition or for binding to MHC. Such modified Tcell epitopes may be considered immunologically equivalent.

Preferably a T cell epitope when presented by MHC and recognized by a Tcell receptor is able to induce in the presence of appropriateco-stimulatory signals, clonal expansion of the T cell carrying the Tcell receptor specifically recognizing the peptide/MHC-complex.

Preferably, a T cell epitope comprises an amino acid sequencesubstantially corresponding to the amino acid sequence of a fragment ofan antigen. Preferably, said fragment of an antigen is an MHC class Iand/or class II presented peptide.

A T cell epitope according to the invention preferably relates to aportion or fragment of an antigen which is capable of stimulating animmune response, preferably a cellular response against the antigen orcells characterized by expression of the antigen and preferably bypresentation of the antigen such as diseased cells, in particular cancercells. Preferably, a T cell epitope is capable of stimulating a cellularresponse against a cell characterized by presentation of an antigen withclass I MHC and preferably is capable of stimulating anantigen-responsive cytotoxic T-lymphocyte (CTL).

“Antigen processing” or “processing” refers to the degradation of apeptide, polypeptide or protein into procession products, which arefragments of said peptide, polypeptide or protein (e.g., the degradationof a polypeptide into peptides) and the association of one or more ofthese fragments (e.g., via binding) with MHC molecules for presentationby cells, preferably antigen presenting cells, to specific T cells.

“Antigen presenting cells” (APC) are cells which present peptidefragments of protein antigens in association with MHC molecules on theircell surface. Some APCs may activate antigen specific T cells.

Professional antigen-presenting cells are very efficient atinternalizing antigen, either by phagocytosis or by receptor-mediatedendocytosis, and then displaying a fragment of the antigen, bound to aclass II MHC molecule, on their membrane. The T cell recognizes andinteracts with the antigen-class II MHC molecule complex on the membraneof the antigen-presenting cell. An additional co-stimulatory signal isthen produced by the antigen-presenting cell, leading to activation ofthe T cell. The expression of co-stimulatory molecules is a definingfeature of professional antigen-presenting cells.

The main types of professional antigen-presenting cells are dendriticcells, which have the broadest range of antigen presentation, and areprobably the most important antigen-presenting cells, macrophages,B-cells, and certain activated epithelial cells. Dendritic cells (DCs)are leukocyte populations that present antigens captured in peripheraltissues to T cells via both MHC class II and I antigen presentationpathways. It is well known that dendritic cells are potent inducers ofimmune responses and the activation of these cells is a critical stepfor the induction of antitumoral immunity. Dendritic cells areconveniently categorized as “immature” and “mature” cells, which can beused as a simple way to discriminate between two well characterizedphenotypes. However, this nomenclature should not be construed toexclude all possible intermediate stages of differentiation. Immaturedendritic cells are characterized as antigen presenting cells with ahigh capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e. g. CD54 and CD11) and costimulatory molecules (e. g., CD40, CD80,CD86 and 4-1 BB). Dendritic cell maturation is referred to as the statusof dendritic cell activation at which such antigen-presenting dendriticcells lead to T cell priming, while presentation by immature dendriticcells results in tolerance. Dendritic cell maturation is chiefly causedby biomolecules with microbial features detected by innate receptors(bacterial DNA, viral RNA, endotoxin, etc.), pro-inflammatory cytokines(TNF, IL-1, IFNs), ligation of CD40 on the dendritic cell surface byCD40L, and substances released from cells undergoing stressful celldeath. The dendritic cells can be derived by culturing bone marrow cellsin vitro with cytokines, such as granulocyte-macrophagecolony-stimulating factor (GM-CSF) and tumor necrosis factor alpha.

Non-professional antigen-presenting cells do not constitutively expressthe MHC class II proteins required for interaction with naive T cells;these are expressed only upon stimulation of the non-professionalantigen-presenting cells by certain cytokines such as IFNγ.

Antigen presenting cells can be loaded with MHC class I presentedpeptides by transducing the cells with nucleic acid, preferably RNA,encoding a peptide or polypeptide comprising the peptide to bepresented, e.g. a nucleic acid encoding an antigen or polypeptide usedfor vaccination.

In some embodiments, a pharmaceutical composition or vaccine comprisinga nucleic acid delivery vehicle that targets a dendritic or otherantigen presenting cell may be administered to a patient, resulting intransfection that occurs in vivo. In vivo transfection of dendriticcells, for example, may generally be performed using any methods knownin the art, such as those described in WO 97/24447, or the gene gunapproach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.

According to the invention, the term “antigen presenting cell” alsoincludes target cells.

“Target cell” shall mean a cell which is a target for an immune responsesuch as a cellular immune response. Target cells include cells thatpresent an antigen, i.e. a peptide fragment derived from an antigen, andinclude any undesirable cell such as a cancer cell. In preferredembodiments, the target cell is a cell expressing an antigen asdescribed herein and preferably presenting said antigen with class IMHC.

The term “portion” refers to a fraction. With respect to a particularstructure such as an amino acid sequence or protein the term “portion”thereof may designate a continuous or a discontinuous fraction of saidstructure. Preferably, a portion of an amino acid sequence comprises atleast 1%, at least 5%, at least 10%, at least 20%, at least 30%,preferably at least 40%, preferably at least 50%, more preferably atleast 60%, more preferably at least 70%, even more preferably at least80%, and most preferably at least 90% of the amino acids of said aminoacid sequence. Preferably, if the portion is a discontinuous fractionsaid discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or moreparts of a structure, each part being a continuous element of thestructure. For example, a discontinuous fraction of an amino acidsequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably notmore than 4 parts of said amino acid sequence, wherein each partpreferably comprises at least 5 continuous amino acids, at least 10continuous amino acids, preferably at least 20 continuous amino acids,preferably at least 30 continuous amino acids of the amino acidsequence.

The terms “part” and “fragment” are used interchangeably herein andrefer to a continuous element. For example, a part of a structure suchas an amino acid sequence or protein refers to a continuous element ofsaid structure. A portion, a part or a fragment of a structurepreferably comprises one or more functional properties of saidstructure. For example, a portion, a part or a fragment of an epitope,peptide or protein is preferably immunologically equivalent to theepitope, peptide or protein it is derived from. In the context of thepresent invention, a “part” of a structure such as an amino acidsequence preferably comprises, preferably consists of at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 98%, at least 99% of the entirestructure or amino acid sequence.

The term “immunoreactive cell” in the context of the present inventionrelates to a cell which exerts effector functions during an immunereaction. An “immunoreactive cell” preferably is capable of binding anantigen or a cell characterized by presentation of an antigen or apeptide fragment thereof (e.g. a T cell epitope) and mediating an immuneresponse. For example, such cells secrete cytokines and/or chemokines,secrete antibodies, recognize cancerous cells, and optionally eliminatesuch cells. For example, immunoreactive cells comprise T cells(cytotoxic T cells, helper T cells, tumor infiltrating T cells), Bcells, natural killer cells, neutrophils, macrophages, and dendriticcells. Preferably, in the context of the present invention,“immunoreactive cells” are T cells, preferably CD4⁺ and/or CD8⁺ T cells.

Preferably, an “immunoreactive cell” recognizes an antigen or a peptidefragment thereof with some degree of specificity, in particular ifpresented in the context of MHC molecules such as on the surface ofantigen presenting cells or diseased cells such as cancer cells.Preferably, said recognition enables the cell that recognizes an antigenor a peptide fragment thereof to be responsive or reactive. If the cellis a helper T cell (CD4⁺ T cell) bearing receptors that recognize anantigen or a peptide fragment thereof in the context of MHC class IImolecules such responsiveness or reactivity may involve the release ofcytokines and/or the activation of CD8⁺ lymphocytes (CTLs) and/orB-cells. If the cell is a CTL such responsiveness or reactivity mayinvolve the elimination of cells presented in the context of MHC class Imolecules, i.e., cells characterized by presentation of an antigen withclass I MHC, for example, via apoptosis or perforin-mediated cell lysis.According to the invention, CTL responsiveness may include sustainedcalcium flux, cell division, production of cytokines such as IFN-γ andTNF-α, up-regulation of activation markers such as CD44 and CD69, andspecific cytolytic killing of antigen expressing target cells. CTLresponsiveness may also be determined using an artificial reporter thataccurately indicates CTL responsiveness. Such CTL that recognize anantigen or an antigen fragment and are responsive or reactive are alsotermed “antigen-responsive CTL” herein. If the cell is a B cell suchresponsiveness may involve the release of immunoglobulins.

The terms “T cell” and “T lymphocyte” are used interchangeably hereinand include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs,CD8+ T cells) which comprise cytolytic T cells.

T cells belong to a group of white blood cells known as lymphocytes, andplay a central role in cell-mediated immunity. They can be distinguishedfrom other lymphocyte types, such as B cells and natural killer cells bythe presence of a special receptor on their cell surface called T cellreceptor (TCR). The thymus is the principal organ responsible for thematuration of T cells. Several different subsets of T cells have beendiscovered, each with a distinct function.

T helper cells assist other white blood cells in immunologic processes,including maturation of B cells into plasma cells and activation ofcytotoxic T cells and macrophages, among other functions. These cellsare also known as CD4+ T cells because they express the CD4 protein ontheir surface. Helper T cells become activated when they are presentedwith peptide antigens by MHC class II molecules that are expressed onthe surface of antigen presenting cells (APCs). Once activated, theydivide rapidly and secrete small proteins called cytokines that regulateor assist in the active immune response.

Cytotoxic T cells destroy virally infected cells and tumor cells, andare also implicated in transplant rejection. These cells are also knownas CD8+ T cells since they express the CD8 glycoprotein at theirsurface. These cells recognize their targets by binding to antigenassociated with MHC class I, which is present on the surface of nearlyevery cell of the body.

A majority of T cells have a T cell receptor (TCR) existing as a complexof several proteins. The actual T cell receptor is composed of twoseparate peptide chains, which are produced from the independent T cellreceptor alpha and beta (TCRα and TCRβ) genes and are called α- andβ-TCR chains. γδ T cells (gamma delta T cells) represent a small subsetof T cells that possess a distinct T cell receptor (TCR) on theirsurface. However, in γδ T cells, the TCR is made up of one γ-chain andone δ-chain. This group of T cells is much less common (2% of total Tcells) than the αβ T cells.

The first signal in activation of T cells is provided by binding of theT cell receptor to a short peptide presented by the MHC on another cell.This ensures that only a T cell with a TCR specific to that peptide isactivated. The partner cell is usually an antigen presenting cell suchas a professional antigen presenting cell, usually a dendritic cell inthe case of naïve responses, although B cells and macrophages can beimportant APCs.

T cells described herein may also comprise an artificial T cellreceptor, e.g. instead of or in addition to the T cell's own T cellreceptor. Such T cells do not necessarily require processing andpresentation of an antigen for recognition of the target cell but rathermay recognize preferably with specificity any antigen present on atarget cell. Preferably, said artificial T cell receptor is expressed onthe surface of the cells. For the purpose of the present invention Tcells comprising an artificial T cell receptor are comprised by the term“T cell” as used herein.

According to the invention the term “artificial T cell receptor” issynonymous with the terms “chimeric T cell receptor” and “chimericantigen receptor (CAR)”. These terms relate to engineered receptors,which confer an arbitrary specificity such as the specificity of amonoclonal antibody onto an immune effector cell such as a T cell. Inthis way, a large number of cancer-specific T cells can be generated foradoptive cell transfer.

In one embodiment, a single-chain variable fragment (scFv) derived froma monoclonal antibody is fused to CD3-zeta transmembrane and endodomain.Such molecules result in the transmission of a zeta signal in responseto recognition by the scFv of its antigen target on a target cell andkilling of the target cell that expresses the target antigen. Antigenrecognition domains which also may be used include among others T-cellreceptor (TCR) alpha and beta single chains. In fact almost anythingthat binds a given target with high affinity can be used as an antigenrecognition domain.

Following antigen recognition, receptors cluster and a signal istransmitted to the cell. The most commonly used endodomain component isCD3-zeta. This transmits an activation signal to the T cell afterantigen is bound.

Adoptive cell transfer therapy with CAR-engineered T cells expressingchimeric antigen receptors is a promising anti-cancer therapeutic asCAR-modified T cells can be engineered to target virtually any tumorantigen. For example, patient's T cells may be genetically engineered toexpress CARs specifically directed towards antigens on the patient'stumor cells, then infused back into the patient.

According to the present invention, a molecule is capable of binding toa target if it has a significant affinity for said predetermined targetand binds to said predetermined target in standard assays. “Affinity” or“binding affinity” is often measured by equilibrium dissociationconstant (K_(D)). A molecule is not (substantially) capable of bindingto a target if it has no significant affinity for said target and doesnot bind significantly to said target in standard assays.

Cytotoxic T lymphocytes may be generated in vivo by incorporation of anantigen, an immunogenic peptide fragment thereof or polypeptidecomprising such immunogenic peptide fragment into antigen-presentingcells in vivo. The antigen, peptide fragment or polypeptide may berepresented as protein, as DNA (e.g. within a vector) or as RNA. Theantigen or polypeptide may be processed to produce a peptide partner forthe MHC molecule, while a fragment thereof may be presented without theneed for further processing. The latter is the case in particular, ifthese can bind to MHC molecules. In general, administration to a patientby intradermal injection is possible. However, injection may also becarried out intranodally into a lymph node (Maloy et al. (2001), ProcNatl Acad Sci USA 98:3299-303). The resulting cells present the complexof interest and are recognized by autologous cytotoxic T lymphocyteswhich then propagate.

Specific activation of CD4+ or CD8+ T cells may be detected in a varietyof ways. Methods for detecting specific T cell activation includedetecting the proliferation of T cells, the production of cytokines(e.g., lymphokines), or the generation of cytolytic activity. For CD4+ Tcells, a preferred method for detecting specific T cell activation isthe detection of the proliferation of T cells. For CD8+ T cells, apreferred method for detecting specific T cell activation is thedetection of the generation of cytolytic activity.

By “cell characterized by presentation of an antigen” or “cellpresenting an antigen” or similar expressions is meant a cell such as adiseased cell, e.g. a cancer cell, or an antigen presenting cellpresenting the antigen it expresses or a fragment derived from saidantigen, e.g. by processing of the antigen, in the context of MHCmolecules, in particular MHC Class I molecules. Similarly, the terms“disease characterized by presentation of an antigen” denotes a diseaseinvolving cells characterized by presentation of an antigen, inparticular with class I MHC. Presentation of an antigen by a cell may beeffected by transfecting the cell with a nucleic acid such as RNAencoding the antigen.

By “fragment of an antigen which is presented” or similar expressions ismeant that the fragment can be presented by MHC class I or class II,preferably MHC class I, e.g. when added directly to antigen presentingcells. In one embodiment, the fragment is a fragment which is naturallypresented by cells expressing an antigen.

The term “immunologically equivalent” means that the immunologicallyequivalent molecule such as the immunologically equivalent amino acidsequence exhibits the same or essentially the same immunologicalproperties and/or exerts the same or essentially the same immunologicaleffects, e.g., with respect to the type of the immunological effect suchas induction of a humoral and/or cellular immune response, the strengthand/or duration of the induced immune reaction, or the specificity ofthe induced immune reaction. In the context of the present invention,the term “immunologically equivalent” is preferably used with respect tothe immunological effects or properties of a peptide used forimmunization. For example, an amino acid sequence is immunologicallyequivalent to a reference amino acid sequence if said amino acidsequence when exposed to the immune system of a subject induces animmune reaction having a specificity of reacting with the referenceamino acid sequence.

The term “immune effector functions” in the context of the presentinvention includes any functions mediated by components of the immunesystem that result, for example, in the killing of tumor cells, or inthe inhibition of tumor growth and/or inhibition of tumor development,including inhibition of tumor dissemination and metastasis. Preferably,the immune effector functions in the context of the present inventionare T cell mediated effector functions. Such functions comprise in thecase of a helper T cell (CD4⁺ T cell) the recognition of an antigen oran antigen fragment in the context of MHC class II molecules by T cellreceptors, the release of cytokines and/or the activation of CD8⁺lymphocytes (CTLs) and/or B-cells, and in the case of CTL therecognition of an antigen or an antigen fragment in the context of MHCclass I molecules by T cell receptors, the elimination of cellspresented in the context of MHC class I molecules, i.e., cellscharacterized by presentation of an antigen with class I MHC, forexample, via apoptosis or perforin-mediated cell lysis, production ofcytokines such as IFN-γ and TNF-α, and specific cytolytic killing ofantigen expressing target cells.

According to the invention, epitopes may have amino acid modificationswhich may result from mutations in the nucleic acid of a cell. Suchmutations may be identified by known sequencing techniques.

In one embodiment, the mutations are cancer specific somatic mutationsin a tumor specimen of a cancer patient which may be determined byidentifying sequence differences between the genome, exome and/ortranscriptome of a tumor specimen and the genome, exome and/ortranscriptome of a non-tumorigenous specimen.

According to the invention a tumor specimen relates to any sample suchas a bodily sample derived from a patient containing or being expectedof containing tumor or cancer cells. The bodily sample may be any tissuesample such as blood, a tissue sample obtained from the primary tumor orfrom tumor metastases or any other sample containing tumor or cancercells. Preferably, a bodily sample is blood and cancer specific somaticmutations or sequence differences are determined in one or morecirculating tumor cells (CTCs) contained in the blood. In anotherembodiment, a tumor specimen relates to one or more isolated tumor orcancer cells such as circulating tumor cells (CTCs) or a samplecontaining one or more isolated tumor or cancer cells such ascirculating tumor cells (CTCs).

A non-tumorigenous specimen relates to any sample such as a bodilysample derived from a patient or another individual which preferably isof the same species as the patient, preferably a healthy individual notcontaining or not being expected of containing tumor or cancer cells.The bodily sample may be any tissue sample such as blood or a samplefrom a non-tumorigenous tissue.

The invention may involve the determination of the cancer mutationsignature of a patient. The term “cancer mutation signature” may referto all cancer mutations present in one or more cancer cells of a patientor it may refer to only a portion of the cancer mutations present in oneor more cancer cells of a patient. Accordingly, the present inventionmay involve the identification of all cancer specific mutations presentin one or more cancer cells of a patient or it may involve theidentification of only a portion of the cancer specific mutationspresent in one or more cancer cells of a patient.

Preferably, the mutations identified according to the present inventionare non-synonymous mutations, preferably non-synonymous mutations ofproteins expressed in a tumor or cancer cell.

In one embodiment, cancer specific somatic mutations or sequencedifferences are determined in the genome, preferably the entire genome,of a tumor specimen. Thus, the invention may comprise identifying thecancer mutation signature of the genome, preferably the entire genome ofone or more cancer cells. In one embodiment, the step of identifyingcancer specific somatic mutations in a tumor specimen of a cancerpatient comprises identifying the genome-wide cancer mutation profile.

In one embodiment, cancer specific somatic mutations or sequencedifferences are determined in the exome, preferably the entire exome, ofa tumor specimen. Thus, the invention may comprise identifying thecancer mutation signature of the exome, preferably the entire exome ofone or more cancer cells. In one embodiment, the step of identifyingcancer specific somatic mutations in a tumor specimen of a cancerpatient comprises identifying the exome-wide cancer mutation profile.

In one embodiment, cancer specific somatic mutations or sequencedifferences are determined in the transcriptome, preferably the entiretranscriptome, of a tumor specimen. Thus, the invention may compriseidentifying the cancer mutation signature of the transcriptome,preferably the entire transcriptome of one or more cancer cells. In oneembodiment, the step of identifying cancer specific somatic mutations ina tumor specimen of a cancer patient comprises identifying thetranscriptome-wide cancer mutation profile.

In one embodiment, the step of identifying cancer specific somaticmutations or identifying sequence differences comprises single cellsequencing of one or more, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or even more cancer cells. Thus, theinvention may comprise identifying a cancer mutation signature of saidone or more cancer cells. In one embodiment, the cancer cells arecirculating tumor cells. The cancer cells such as the circulating tumorcells may be isolated prior to single cell sequencing.

In one embodiment, the step of identifying cancer specific somaticmutations or identifying sequence differences involves using nextgeneration sequencing (NGS).

In one embodiment, the step of identifying cancer specific somaticmutations or identifying sequence differences comprises sequencinggenomic DNA and/or RNA of the tumor specimen.

To reveal cancer specific somatic mutations or sequence differences thesequence information obtained from the tumor specimen is preferablycompared with a reference such as sequence information obtained fromsequencing nucleic acid such as DNA or RNA of normal non-cancerous cellssuch as germline cells which may either be obtained from the patient ora different individual. In one embodiment, normal genomic germline DNAis obtained from peripheral blood mononuclear cells (PBMCs)

The term “genome” relates to the total amount of genetic information inthe chromosomes of an organism or a cell.

The term “exome” refers to part of the genome of an organism formed byexons, which are coding portions of expressed genes. The exome providesthe genetic blueprint used in the synthesis of proteins and otherfunctional gene products. It is the most functionally relevant part ofthe genome and, therefore, it is most likely to contribute to thephenotype of an organism. The exome of the human genome is estimated tocomprise 1.5% of the total genome (Ng, P C et al., PLoS Gen., 4(8):1-15, 2008).

The term “transcriptome” relates to the set of all RNA molecules,including mRNA, r RNA, tRNA, and other non-coding RNA produced in onecell or a population of cells. In context of the present invention thetranscriptome means the set of all RNA molecules produced in one cell, apopulation of cells, preferably a population of cancer cells, or allcells of a given individual at a certain time point.

A “nucleic acid” is according to the invention preferablydeoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferablyRNA, most preferably in vitro transcribed RNA (IVT RNA) or syntheticRNA. Nucleic acids include according to the invention genomic DNA, cDNA,mRNA, recombinantly produced and chemically synthesized molecules.According to the invention, a nucleic acid may be present as asingle-stranded or double-stranded and linear or covalently circularlyclosed molecule. A nucleic acid can, according to the invention, beisolated. The term “isolated nucleic acid” means, according to theinvention, that the nucleic acid (i) was amplified in vitro, for examplevia polymerase chain reaction (PCR), (ii) was produced recombinantly bycloning, (iii) was purified, for example, by cleavage and separation bygel electrophoresis, or (iv) was synthesized, for example, by chemicalsynthesis. A nucleic can be employed for introduction into, i.e.transfection of, cells, in particular, in the form of RNA which can beprepared by in vitro transcription from a DNA template. The RNA canmoreover be modified before application by stabilizing sequences,capping, and polyadenylation.

The term “genetic material” includes isolated nucleic acid, either DNAor RNA, a section of a double helix, a section of a chromosome, or anorganism's or cell's entire genome, in particular its exome ortranscriptome.

The term “mutation” refers to a change of or difference in the nucleicacid sequence (nucleotide substitution, addition or deletion) comparedto a reference. A “somatic mutation” can occur in any of the cells ofthe body except the germ cells (sperm and egg) and therefore are notpassed on to children. These alterations can (but do not always) causecancer or other diseases. Preferably a mutation is a non-synonymousmutation. The term “non-synonymous mutation” refers to a mutation,preferably a nucleotide substitution, which does result in an amino acidchange such as an amino acid substitution in the translation product.

According to the invention, the term “mutation” includes pointmutations, Indels, fusions, chromothripsis and RNA edits.

According to the invention, the term “Indel” describes a specialmutation class, defined as a mutation resulting in a colocalizedinsertion and deletion and a net gain or loss in nucleotides. In codingregions of the genome, unless the length of an indel is a multiple of 3,they produce a frameshift mutation. Indels can be contrasted with apoint mutation; where an Indel inserts and deletes nucleotides from asequence, a point mutation is a form of substitution that replaces oneof the nucleotides.

Fusions can generate hybrid genes formed from two previously separategenes. It can occur as the result of a translocation, interstitialdeletion, or chromosomal inversion. Often, fusion genes are oncogenes.Oncogenic fusion genes may lead to a gene product with a new ordifferent function from the two fusion partners. Alternatively, aproto-oncogene is fused to a strong promoter, and thereby the oncogenicfunction is set to function by an upregulation caused by the strongpromoter of the upstream fusion partner. Oncogenic fusion transcriptsmay also be caused by trans-splicing or read-through events.

According to the invention, the term “chromothripsis” refers to agenetic phenomenon by which specific regions of the genome are shatteredand then stitched together via a single devastating event.

According to the invention, the term “RNA edit” or “RNA editing” refersto molecular processes in which the information content in an RNAmolecule is altered through a chemical change in the base makeup. RNAediting includes nucleoside modifications such as cytidine (C) touridine (U) and adenosine (A) to inosine (I) deaminations, as well asnon-templated nucleotide additions and insertions. RNA editing in mRNAseffectively alters the amino acid sequence of the encoded protein sothat it differs from that predicted by the genomic DNA sequence.

The term “cancer mutation signature” refers to a set of mutations whichare present in cancer cells when compared to non-cancerous referencecells.

According to the invention, a “reference” may be used to correlate andcompare the results obtained from a tumor specimen. Typically the“reference” may be obtained on the basis of one or more normalspecimens, in particular specimens which are not affected by a cancerdisease, either obtained from a patient or one or more differentindividuals, preferably healthy individuals, in particular individualsof the same species. A “reference” can be determined empirically bytesting a sufficiently large number of normal specimens.

In the context of the present invention, the term “RNA” relates to amolecule which comprises ribonucleotide residues and preferably beingentirely or substantially composed of ribonucleotide residues.“Ribonucleotide” relates to a nucleotide with a hydroxyl group at the2′-position of a β-D-ribofuranosyl group. The term “RNA” comprisesdouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallyor completely purified RNA, essentially pure RNA, synthetic RNA, andrecombinantly generated RNA such as modified RNA which differs fromnaturally occurring RNA by addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of a RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in RNA molecules can also comprise non-standard nucleotides,such as non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs or analogs of naturally-occurring RNA.

According to the present invention, the term “RNA” includes andpreferably relates to “mRNA”. The term “mRNA” means “messenger-RNA” andrelates to a “transcript” which is generated by using a DNA template andencodes a peptide or polypeptide. Typically, an mRNA comprises a 5′-UTR,a protein coding region, and a 3′-UTR. mRNA only possesses limitedhalf-life in cells and in vitro. In the context of the presentinvention, mRNA may be generated by in vitro transcription from a DNAtemplate. The in vitro transcription methodology is known to the skilledperson. For example, there is a variety of in vitro transcription kitscommercially available.

According to the invention, the stability and translation efficiency ofRNA may be modified as required. For example, RNA may be stabilized andits translation increased by one or more modifications having astabilizing effects and/or increasing translation efficiency of RNA.Such modifications are described, for example, in PCT/EP2006/009448incorporated herein by reference. In order to increase expression of theRNA used according to the present invention, it may be modified withinthe coding region, i.e. the sequence encoding the expressed peptide orprotein, preferably without altering the sequence of the expressedpeptide or protein, so as to increase the GC-content to increase mRNAstability and to perform a codon optimization and, thus, enhancetranslation in cells.

The term “modification” in the context of the RNA used in the presentinvention includes any modification of an RNA which is not naturallypresent in said RNA.

In one embodiment of the invention, the RNA used according to theinvention does not have uncapped 5′-triphosphates. Removal of suchuncapped 5′-triphosphates can be achieved by treating RNA with aphosphatase.

The RNA according to the invention may have modified ribonucleotides inorder to increase its stability and/or decrease cytotoxicity. Forexample, in one embodiment, in the RNA used according to the invention5-methylcytidine is substituted partially or completely, preferablycompletely, for cytidine. Alternatively or additionally, in oneembodiment, in the RNA used according to the invention pseudouridine issubstituted partially or completely, preferably completely, for uridine.

In one embodiment, the term “modification” relates to providing an RNAwith a 5′-cap or 5′-cap analog. The term “5′-cap” refers to a capstructure found on the 5′-end of an mRNA molecule and generally consistsof a guanosine nucleotide connected to the mRNA via an unusual 5′ to 5′triphosphate linkage. In one embodiment, this guanosine is methylated atthe 7-position. The term “conventional 5′-cap” refers to a naturallyoccurring RNA 5′-cap, preferably to the 7-methylguanosine cap (m⁷G). Inthe context of the present invention, the term “5′-cap” includes a5′-cap analog that resembles the RNA cap structure and is modified topossess the ability to stabilize RNA and/or enhance translation of RNAif attached thereto, preferably in vivo and/or in a cell.

Providing an RNA with a 5′-cap or 5′-cap analog may be achieved by invitro transcription of a DNA template in presence of said 5′-cap or5′-cap analog, wherein said 5′-cap is co-transcriptionally incorporatedinto the generated RNA strand, or the RNA may be generated, for example,by in vitro transcription, and the 5′-cap may be attached to the RNApost-transcriptionally using capping enzymes, for example, cappingenzymes of vaccinia virus.

The RNA may comprise further modifications. For example, a furthermodification of the RNA used in the present invention may be anextension or truncation of the naturally occurring poly(A) tail or analteration of the 5′- or 3′-untranslated regions (UTR) such asintroduction of a UTR which is not related to the coding region of saidRNA, for example, the exchange of the existing 3′-UTR with or theinsertion of one or more, preferably two copies of a 3′-UTR derived froma globin gene, such as alpha2-globin, alpha1-globin, beta-globin,preferably beta-globin, more preferably human beta-globin.

RNA having an unmasked poly-A sequence is translated more efficientlythan RNA having a masked poly-A sequence. The term “poly(A) tail” or“poly-A sequence” relates to a sequence of adenyl (A) residues whichtypically is located on the 3′-end of a RNA molecule and “unmaskedpoly-A sequence” means that the poly-A sequence at the 3′ end of an RNAmolecule ends with an A of the poly-A sequence and is not followed bynucleotides other than A located at the 3′ end, i.e. downstream, of thepoly-A sequence. Furthermore, a long poly-A sequence of about 120 basepairs results in an optimal transcript stability and translationefficiency of RNA.

Therefore, in order to increase stability and/or expression of the RNAused according to the present invention, it may be modified so as to bepresent in conjunction with a poly-A sequence, preferably having alength of 10 to 500, more preferably 30 to 300, even more preferably 65to 200 and especially 100 to 150 adenosine residues. In an especiallypreferred embodiment the poly-A sequence has a length of approximately120 adenosine residues. To further increase stability and/or expressionof the RNA used according to the invention, the poly-A sequence can beunmasked.

In addition, incorporation of a 3′-non translated region (UTR) into the3′-non translated region of an RNA molecule can result in an enhancementin translation efficiency. A synergistic effect may be achieved byincorporating two or more of such 3′-non translated regions. The 3′-nontranslated regions may be autologous or heterologous to the RNA intowhich they are introduced. In one particular embodiment the 3′-nontranslated region is derived from the human β-globin gene.

A combination of the above described modifications, i.e. incorporationof a poly-A sequence, unmasking of a poly-A sequence and incorporationof one or more 3′-non translated regions, has a synergistic influence onthe stability of RNA and increase in translation efficiency.

The term “stability” of RNA relates to the “half-life” of RNA.“Half-life” relates to the period of time which is needed to eliminatehalf of the activity, amount, or number of molecules. In the context ofthe present invention, the half-life of an RNA is indicative for thestability of said RNA. The half-life of RNA may influence the “durationof expression” of the RNA. It can be expected that RNA having a longhalf-life will be expressed for an extended time period.

Of course, if according to the present invention it is desired todecrease stability and/or translation efficiency of RNA, it is possibleto modify RNA so as to interfere with the function of elements asdescribed above increasing the stability and/or translation efficiencyof RNA.

The term “expression” is used according to the invention in its mostgeneral meaning and comprises the production of RNA and/or peptides,polypeptides or proteins, e.g. by transcription and/or translation. Withrespect to RNA, the term “expression” or “translation” relates inparticular to the production of peptides, polypeptides or proteins. Italso comprises partial expression of nucleic acids. Moreover, expressioncan be transient or stable. According to the invention, a tumor antigenis expressed in a cell if the tumor antigen can be detected in the cellor a lysate thereof by conventional techniques for protein detectionsuch as techniques using antibodies specifically binding to the tumorantigen. Preferably, a tumor antigen is expressed in a cell if T cellsare able to bind to a peptide fragment derived from the tumor antigenpresented on the cell in the context of MHC molecules and preferably areable to exert immune effector functions on the cell.

According to the invention, the term expression also includes an“aberrant expression” or “abnormal expression”. “Aberrant expression” or“abnormal expression” means according to the invention that expressionis altered, preferably increased, compared to a reference, e.g. a statein a subject not having a disease associated with aberrant or abnormalexpression of a certain protein, e.g., a tumor antigen. An increase inexpression refers to an increase by at least 10%, in particular at least20%, at least 50% or at least 100%, or more. In one embodiment,expression is only found in a diseased tissue, while expression in ahealthy tissue is repressed.

The term “specifically expressed” means that a protein is essentiallyonly expressed in a specific tissue or organ. For example, a tumorantigen specifically expressed in gastric mucosa means that said proteinis primarily expressed in gastric mucosa and is not expressed in othertissues or is not expressed to a significant extent in other tissue ororgan types. Thus, a protein that is exclusively expressed in cells ofthe gastric mucosa and to a significantly lesser extent in any othertissue, such as testis, is specifically expressed in cells of thegastric mucosa. In some embodiments, a tumor antigen may also bespecifically expressed under normal conditions in more than one tissuetype or organ, such as in 2 or 3 tissue types or organs, but preferablyin not more than 3 different tissue or organ types. In this case, thetumor antigen is then specifically expressed in these organs. Forexample, if a tumor antigen is expressed under normal conditionspreferably to an approximately equal extent in lung and stomach, saidtumor antigen is specifically expressed in lung and stomach.

In the context of the present invention, the term “transcription”relates to a process, wherein the genetic code in a DNA sequence istranscribed into RNA. Subsequently, the RNA may be translated intoprotein. According to the present invention, the term “transcription”comprises “in vitro transcription”, wherein the term “in vitrotranscription” relates to a process wherein RNA, in particular mRNA, isin vitro synthesized in a cell-free system, preferably using appropriatecell extracts. Preferably, cloning vectors are applied for thegeneration of transcripts. These cloning vectors are generallydesignated as transcription vectors and are according to the presentinvention encompassed by the term “vector”. According to the presentinvention, the RNA used in the present invention preferably is in vitrotranscribed RNA (IVT-RNA) and may be obtained by in vitro transcriptionof an appropriate DNA template. The promoter for controllingtranscription can be any promoter for any RNA polymerase. Particularexamples of RNA polymerases are the T7, T3, and SP6 RNA polymerases.Preferably, the in vitro transcription according to the invention iscontrolled by a T7 or SP6 promoter. A DNA template for in vitrotranscription may be obtained by cloning of a nucleic acid, inparticular cDNA, and introducing it into an appropriate vector for invitro transcription. The cDNA may be obtained by reverse transcriptionof RNA.

The term “translation” according to the invention relates to the processin the ribosomes of a cell by which a strand of messenger RNA directsthe assembly of a sequence of amino acids to make a peptide, polypeptideor protein.

Expression control sequences or regulatory sequences, which according tothe invention may be linked functionally with a nucleic acid, can behomologous or heterologous with respect to the nucleic acid. A codingsequence and a regulatory sequence are linked together “functionally” ifthey are bound together covalently, so that the transcription ortranslation of the coding sequence is under the control or under theinfluence of the regulatory sequence. If the coding sequence is to betranslated into a functional protein, with functional linkage of aregulatory sequence with the coding sequence, induction of theregulatory sequence leads to a transcription of the coding sequence,without causing a reading frame shift in the coding sequence orinability of the coding sequence to be translated into the desiredprotein or peptide.

The term “expression control sequence” or “regulatory sequence”comprises, according to the invention, promoters, ribosome-bindingsequences and other control elements, which control the transcription ofa nucleic acid or the translation of the derived RNA. In certainembodiments of the invention, the regulatory sequences can becontrolled. The precise structure of regulatory sequences can varydepending on the species or depending on the cell type, but generallycomprises 5′-untranscribed and 5′- and 3′-untranslated sequences, whichare involved in the initiation of transcription or translation, such asTATA-box, capping-sequence, CAAT-sequence and the like. In particular,5′ untranscribed regulatory sequences comprise a promoter region thatincludes a promoter sequence for transcriptional control of thefunctionally bound gene. Regulatory sequences can also comprise enhancersequences or upstream activator sequences.

Preferably, according to the invention, RNA to be expressed in a cell isintroduced into said cell. In one embodiment of the methods according tothe invention, the RNA that is to be introduced into a cell is obtainedby in vitro transcription of an appropriate DNA template.

According to the invention, terms such as “RNA capable of expressing”and “RNA encoding” are used interchangeably herein and with respect to aparticular peptide or polypeptide mean that the RNA, if present in theappropriate environment, preferably within a cell, can be expressed toproduce said peptide or polypeptide. Preferably, RNA according to theinvention is able to interact with the cellular translation machinery toprovide the peptide or polypeptide it is capable of expressing.

Terms such as “transferring”, “introducing” or “transfecting” are usedinterchangeably herein and relate to the introduction of nucleic acids,in particular exogenous or heterologous nucleic acids, in particular RNAinto a cell. According to the present invention, the cell can form partof an organ, a tissue and/or an organism. According to the presentinvention, the administration of a nucleic acid is either achieved asnaked nucleic acid or in combination with an administration reagent.Preferably, administration of nucleic acids is in the form of nakednucleic acids. Preferably, the RNA is administered in combination withstabilizing substances such as RNase inhibitors. The present inventionalso envisions the repeated introduction of nucleic acids into cells toallow sustained expression for extended time periods.

Cells can be transfected with any carriers with which RNA can beassociated, e.g. by forming complexes with the RNA or forming vesiclesin which the RNA is enclosed or encapsulated, resulting in increasedstability of the RNA compared to naked RNA. Carriers useful according tothe invention include, for example, lipid-containing carriers such ascationic lipids, liposomes, in particular cationic liposomes, andmicelles, and nanoparticles. Cationic lipids may form complexes withnegatively charged nucleic acids. Any cationic lipid may be usedaccording to the invention.

Preferably, the introduction of RNA which encodes a peptide orpolypeptide into a cell, in particular into a cell present in vivo,results in expression of said peptide or polypeptide in the cell. Inparticular embodiments, the targeting of the nucleic acids to particularcells is preferred. In such embodiments, a carrier which is applied forthe administration of the nucleic acid to a cell (for example, aretrovirus or a liposome), exhibits a targeting molecule. For example, amolecule such as an antibody which is specific for a surface membraneprotein on the target cell or a ligand for a receptor on the target cellmay be incorporated into the nucleic acid carrier or may be boundthereto. In case the nucleic acid is administered by liposomes, proteinswhich bind to a surface membrane protein which is associated withendocytosis may be incorporated into the liposome formulation in orderto enable targeting and/or uptake. Such proteins encompass capsidproteins of fragments thereof which are specific for a particular celltype, antibodies against proteins which are internalized, proteins whichtarget an intracellular location etc.

The term “cell” or “host cell” preferably is an intact cell, i.e. a cellwith an intact membrane that has not released its normal intracellularcomponents such as enzymes, organelles, or genetic material. An intactcell preferably is a viable cell, i.e. a living cell capable of carryingout its normal metabolic functions. Preferably said term relatesaccording to the invention to any cell which can be transformed ortransfected with an exogenous nucleic acid. The term “cell” includesaccording to the invention prokaryotic cells (e.g., E. coli) oreukaryotic cells (e.g., dendritic cells, B cells, CHO cells, COS cells,K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells).The exogenous nucleic acid may be found inside the cell (i) freelydispersed as such, (ii) incorporated in a recombinant vector, or (iii)integrated into the host cell genome or mitochondrial DNA. Mammaliancells are particularly preferred, such as cells from humans, mice,hamsters, pigs, goats, and primates. The cells may be derived from alarge number of tissue types and include primary cells and cell lines.Specific examples include keratinocytes, peripheral blood leukocytes,bone marrow stem cells, and embryonic stem cells. In furtherembodiments, the cell is an antigen-presenting cell, in particular adendritic cell, a monocyte, or macrophage.

A cell which comprises a nucleic acid molecule preferably expresses thepeptide or polypeptide encoded by the nucleic acid.

The term “clonal expansion” refers to a process wherein a specificentity is multiplied. In the context of the present invention, the termis preferably used in the context of an immunological response in whichlymphocytes are stimulated by an antigen, proliferate, and the specificlymphocyte recognizing said antigen is amplified. Preferably, clonalexpansion leads to differentiation of the lymphocytes.

Terms such as “reducing” or “inhibiting” relate to the ability to causean overall decrease, preferably of 5% or greater, 10% or greater, 20% orgreater, more preferably of 50% or greater, and most preferably of 75%or greater, in the level. The term “inhibit” or similar phrases includesa complete or essentially complete inhibition, i.e. a reduction to zeroor essentially to zero.

Terms such as “increasing”, “enhancing”, “promoting” or “prolonging”preferably relate to an increase, enhancement, promotion or prolongationby about at least 10%, preferably at least 20%, preferably at least 30%,preferably at least 40%, preferably at least 50%, preferably at least80%, preferably at least 100%, preferably at least 200% and inparticular at least 300%. These terms may also relate to an increase,enhancement, promotion or prolongation from zero or a non-measurable ornon-detectable level to a level of more than zero or a level which ismeasurable or detectable.

According to the invention, the term “vaccine” relates to apharmaceutical preparation (pharmaceutical composition) or product thatupon administration induces an immune response, in particular a cellularimmune response, which recognizes and attacks a pathogen or a diseasedcell such as a cancer cell. A vaccine may be used for the prevention ortreatment of a disease. The term “personalized cancer vaccine” or“individualized cancer vaccine” concerns a particular cancer patient andmeans that a cancer vaccine is adapted to the needs or specialcircumstances of an individual cancer patient.

In one embodiment, a vaccine provided according to the invention maycomprise one or more peptides or polypeptides comprising immunogenicepitopes such as T cell epitopes or a nucleic acid, preferably RNA,encoding said peptides or polypeptides.

The cancer vaccines provided according to the invention whenadministered to a patent preferably provide one or more T cell epitopessuitable for stimulating, priming and/or expanding T cells specific forthe patients tumor. The T cells are preferably directed against cellsexpressing antigens from which the T cell epitopes are derived. Thus,the vaccines described herein are preferably capable of inducing orpromoting a cellular response, preferably cytotoxic T cell activity,against a cancer disease characterized by presentation of one or moretumor antigens with class I MHC.

In one embodiment, a vaccine provided according to the invention relatesto a vaccine which when administered to a patent preferably provides oneor more T cell epitopes, such as 2 or more, 5 or more, 10 or more, 15 ormore, 20 or more, 25 or more, 30 or more and preferably up to 60, up to55, up to 50, up to 45, up to 40, up to 35 or up to 30 T cell epitopes.Presentation of these epitopes by cells of a patient, in particularantigen presenting cells, preferably results in T cells targeting theepitopes when bound to MHC and thus, the patient's tumor, preferably theprimary tumor as well as tumor metastases, expressing antigens fromwhich the T cell epitopes are derived and presenting the same epitopeson the surface of the tumor cells.

Epitopes provided by a vaccine of the invention are preferably presentin the form of a polypeptide comprising said epitopes such as apolyepitopic polypeptide or a nucleic acid, in particular RNA, encodingsaid polypeptide. Furthermore, the epitopes may be present in thepolypeptide in the form of a vaccine sequence, i.e. present in theirnatural sequence context, e.g. flanked by amino acid sequences alsoflanking said epitopes in the naturally occurring protein. Such flankingsequences each may comprise 5 or more, 10 or more, 15 or more, 20 ormore and preferably up to 50, up to 45, up to 40, up to 35 or up to 30amino acids and may flank the epitope sequence N-terminally and/orC-terminally. Thus, a vaccine sequence may comprise 20 or more, 25 ormore, 30 or more, 35 or more, 40 or more and preferably up to 50, up to45, up to 40, up to 35 or up to 30 amino acids. In one embodiment, theepitopes and/or vaccine sequences are lined up in the polypeptidehead-to-tail.

In one embodiment, the epitopes and/or vaccine sequences are spaced bylinkers, in particular neutral linkers. The term “linker” according tothe invention relates to a peptide added between two peptide domainssuch as epitopes or vaccine sequences to connect said peptide domains.There is no particular limitation regarding the linker sequence.However, it is preferred that the linker sequence reduces sterichindrance between the two peptide domains, is well translated, andsupports or allows processing of the epitopes. Furthermore, the linkershould have no or only little immunogenic sequence elements. Linkerspreferably should not create non-endogenous epitopes like thosegenerated from the junction suture between adjacent epitopes, whichmight generate unwanted immune reactions. Therefore, the polyepitopicvaccine should preferably contain linker sequences which are able toreduce the number of unwanted MHC binding junction epitopes. Hoyt et al.(EMBO J. 25(8), 1720-9, 2006) and Zhang et al. (J. Biol. Chem., 279(10),8635-41, 2004) have shown that glycine-rich sequences impair proteasomalprocessing and thus the use of glycine rich linker sequences act tominimize the number of linker-contained peptides that can be processedby the proteasome. Furthermore, glycine was observed to inhibit a strongbinding in MHC binding groove positions (Abastado et al., J. Immunol.151(7), 3569-75, 1993). Schlessinger et al. (Proteins, 61(1), 115-26,2005) had found that amino acids glycine and serine included in an aminoacid sequence result in a more flexible protein that is more efficientlytranslated and processed by the proteasome, enabling better access tothe encoded epitopes. The linker each may comprise 3 or more, 6 or more,9 or more, 10 or more, 15 or more, 20 or more and preferably up to 50,up to 45, up to 40, up to 35 or up to 30 amino acids. Preferably thelinker is enriched in glycine and/or serine amino acids. Preferably, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% of the amino acids of the linker are glycine and/or serine. Inone preferred embodiment, a linker is substantially composed of theamino acids glycine and serine. In one embodiment, the linker comprisesthe amino acid sequence (GGS)_(a)(GSS)_(b)(GGG)_(c)(SSG)_(d)(GSG)_(e)wherein a, b, c, d and e is independently a number selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20and wherein a+b+c+d+e are different from 0 and preferably are 2 or more,3 or more, 4 or more or 5 or more. In one embodiment, the linkercomprises the sequence GGSGGGGSG.

In one particularly preferred embodiment, a polypeptide incorporatingone or more epitopes such as a polyepitopic polypeptide according to thepresent invention is administered to a patient in the form of a nucleicacid, preferably RNA such as in vitro transcribed or synthetic RNA,which may be expressed in cells of a patient such as antigen presentingcells to produce the polypeptide. The present invention also envisionsthe administration of one or more multiepitopic polypeptides which forthe purpose of the present invention are comprised by the term“polyepitopic polypeptide”, preferably in the form of a nucleic acid,preferably RNA such as in vitro transcribed or synthetic RNA, which maybe expressed in cells of a patient such as antigen presenting cells toproduce the one or more polypeptides. In the case of an administrationof more than one multiepitopic polypeptide the epitopes provided by thedifferent multiepitopic polypeptides may be different or partiallyoverlapping. Once present in cells of a patient such as antigenpresenting cells the polypeptide according to the invention is processedto produce the epitopes. Administration of a vaccine provided accordingto the invention may provide MHC class II-presented epitopes that arecapable of eliciting a CD4+ helper T cell response against cellsexpressing antigens from which the MHC presented epitopes are derived.Alternatively or additionally, administration of a vaccine providedaccording to the invention may provide MHC class I-presented epitopesthat are capable of eliciting a CD8+ T cell response against cellsexpressing antigens from which the MHC presented epitopes are derived.Furthermore, administration of a vaccine provided according to theinvention may provide one or more neo-epitopes (including knownneo-epitopes and neo-epitopes identified according to the invention) aswell as one or more epitopes not containing cancer specific somaticmutations. In one embodiment, administration of a vaccine providedaccording to the invention provides neo-epitopes that are MHC classII-presented epitopes and/or are capable of eliciting a CD4+ helper Tcell response against cells expressing antigens from which the MHCpresented epitopes are derived as well as epitopes not containingcancer-specific somatic mutations that are MHC class I-presentedepitopes and/or are capable of eliciting a CD8+ T cell response againstcells expressing antigens from which the MHC presented epitopes arederived. In one embodiment, the neo-epitopes and epitopes not containingcancer-specific somatic mutations have a synergistic effect in thetreatment of cancer. Preferably, a vaccine provided according to theinvention is useful for polyepitopic stimulation of cytotoxic and/orhelper T cell responses.

The vaccine provided according to the invention may be a recombinantvaccine.

The term “recombinant” in the context of the present invention means“made through genetic engineering”. Preferably, a “recombinant entity”such as a recombinant polypeptide in the context of the presentinvention is not occurring naturally, and preferably is a result of acombination of entities such as amino acid or nucleic acid sequenceswhich are not combined in nature. For example, a recombinant polypeptidein the context of the present invention may contain several amino acidsequences such as epitopes or vaccine sequences derived from differentproteins such as CXorf61, CAGE1 and/or PRAME or different portions ofthe same protein fused together, e.g., by peptide bonds or appropriatelinkers.

The term “naturally occurring” as used herein refers to the fact that anobject can be found in nature. For example, a peptide or nucleic acidthat is present in an organism (including viruses) and can be isolatedfrom a source in nature and which has not been intentionally modified byman in the laboratory is naturally occurring.

Agents, compositions and methods described herein can be used to treat asubject with a disease, e.g., a disease characterized by the presence ofdiseased cells expressing one or more antigens and presenting a fragmentthereof. Particularly preferred diseases are cancer diseases. Agents,compositions and methods described herein may also be used forimmunization or vaccination to prevent a disease described herein.

According to the invention, the term “disease” refers to anypathological state, including cancer diseases, in particular those formsof cancer diseases described herein.

The term “normal” refers to the healthy state or the conditions in ahealthy subject or tissue, i.e., non-pathological conditions, wherein“healthy” preferably means non-cancerous.

“Disease involving cells expressing an antigen” means according to theinvention that expression of the antigen in cells of a diseased tissueor organ is detected. Expression in cells of a diseased tissue or organmay be increased compared to the state in a healthy tissue or organ. Anincrease refers to an increase by at least 10%, in particular at least20%, at least 50%, at least 100%, at least 200%, at least 500%, at least1000%, at least 10000% or even more. In one embodiment, expression isonly found in a diseased tissue, while expression in a healthy tissue isrepressed. According to the invention, diseases involving or beingassociated with cells expressing an antigen include cancer diseases.

According to the invention, the term “tumor” or “tumor disease” refersto an abnormal growth of cells (called neoplastic cells, tumorigenouscells or tumor cells) preferably forming a swelling or lesion. By “tumorcell” is meant an abnormal cell that grows by a rapid, uncontrolledcellular proliferation and continues to grow after the stimuli thatinitiated the new growth cease. Tumors show partial or complete lack ofstructural organization and functional coordination with the normaltissue, and usually form a distinct mass of tissue, which may be eitherbenign, pre-malignant or malignant.

Cancer (medical term: malignant neoplasm) is a class of diseases inwhich a group of cells display uncontrolled growth (division beyond thenormal limits), invasion (intrusion on and destruction of adjacenttissues), and sometimes metastasis (spread to other locations in thebody via lymph or blood). These three malignant properties of cancersdifferentiate them from benign tumors, which are self-limited, and donot invade or metastasize. Most cancers form a tumor but some, likeleukemia, do not. Malignancy, malignant neoplasm, and malignant tumorare essentially synonymous with cancer.

Neoplasm is an abnormal mass of tissue as a result of neoplasia.Neoplasia (new growth in Greek) is the abnormal proliferation of cells.The growth of the cells exceeds, and is uncoordinated with that of thenormal tissues around it. The growth persists in the same excessivemanner even after cessation of the stimuli, it usually causes a lump ortumor. Neoplasms may be benign, pre-malignant or malignant.

“Growth of a tumor” or “tumor growth” according to the invention relatesto the tendency of a tumor to increase its size and/or to the tendencyof tumor cells to proliferate.

For purposes of the present invention, the terms “cancer” and “cancerdisease” are used interchangeably with the terms “tumor” and “tumordisease”.

Cancers are classified by the type of cell that resembles the tumor and,therefore, the tissue presumed to be the origin of the tumor. These arethe histology and the location, respectively.

The term “cancer” according to the invention comprises carcinomas,adenocarcinomas, blastomas, leukemias, seminomas, melanomas, teratomas,lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer,kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skincancer, cancer of the brain, cervical cancer, intestinal cancer, livercancer, colon cancer, stomach cancer, intestine cancer, head and neckcancer, gastrointestinal cancer, lymph node cancer, esophagus cancer,colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer,breast cancer, prostate cancer, cancer of the uterus, ovarian cancer andlung cancer and the metastases thereof. Examples thereof are lungcarcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas,renal cell carcinomas, cervical carcinomas, or metastases of the cancertypes or tumors described above. The term cancer according to theinvention also comprises cancer metastases and relapse of cancer.

In one particularly preferred embodiment, the term “cancer” according tothe invention relates to “breast cancer”

The term “breast cancer” relates to a type of cancer originating frombreast tissue, most commonly from the inner lining of milk ducts or thelobules that supply the ducts with milk. Cancers originating from ductsare known as ductal carcinomas, while those originating from lobules areknown as lobular carcinomas. Occasionally, breast cancer presents asmetastatic disease. Common sites of metastasis include bone, liver, lungand brain. Breast cancer occurs in humans and other mammals. While theoverwhelming majority of human cases occur in women, male breast cancercan also occur.

Treatment of breast cancer may include surgery, medications (hormonaltherapy and chemotherapy), radiation and/or immunotherapy.

Breast cancer cells may or may not have three important receptors:estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2).A particularly preferred form of breast cancer according to theinvention is triple-negative breast cancer. The term “triple-negativebreast cancer” refers to any breast cancer that does not express or doesnot overexpress the genes for estrogen receptor (ER), progesteronereceptor (PR) and HER2.

The three main groups of medications used for adjuvant breast cancertreatment are hormone blocking therapy, chemotherapy, and monoclonalantibodies.

Hormone Blocking Therapy

ER positive/PR positive breast cancers can be treated with drugs thateither block the receptors, e.g. tamoxifen (Nolvadex), or alternativelyblock the production of estrogen with an aromatase inhibitor, e.g.anastrozole (Arimidex) or letrozole (Femara). Aromatase inhibitors,however, are only suitable for post-menopausal patients. This is becausethe active aromatase in postmenopausal women is different from theprevalent form in premenopausal women, and therefore these agents areineffective in inhibiting the predominant aromatase of premenopausalwomen.

Chemotherapy

Chemotherapy is predominately used for stage 2-4 disease and isparticularly beneficial in ER negative breast cancer. They are given incombinations, usually for 3-6 months. One of the most common treatmentsis cyclophosphamide plus doxorubicin (adriamycin), known as AC. Mostchemotherapy medications work by destroying fast-growing and/orfast-replicating cancer cells either by causing DNA damage uponreplication or other mechanisms; these drugs also damage fast-growingnormal cells where they cause serious side effects. Damage to the heartmuscle is the most dangerous complication of doxorubicin. Sometimes ataxane drug, such as docetaxel, is added, and the regime is then knownas CAT; taxane attacks the microtubules in cancer cells. Another commontreatment, which produces equivalent results, is cyclophosphamide,methotrexate, and fluorouracil (CMF).

Monoclonal Antibodies

Trastuzumab (Herceptin), a monoclonal antibody to HER2, is onlyeffective in patients with HER2 amplification/overexpression.Trastuzumab, however, is expensive, and approximately 2% of patientssuffer significant heart damage. Other monoclonal antibodies are alsoundergoing clinical trials. Between 25 and thirty percent of breastcancers have an amplification of the HER2 gene or overexpression of itsprotein product. Overexpression of this receptor in breast cancer isassociated with increased disease recurrence and worse prognosis.

According to the invention, a “carcinoma” is a malignant tumor derivedfrom epithelial cells. This group represents the most common cancers,including the common forms of breast, prostate, lung and colon cancer.

“Adenocarcinoma” is a cancer that originates in glandular tissue. Thistissue is also part of a larger tissue category known as epithelialtissue. Epithelial tissue includes skin, glands and a variety of othertissue that lines the cavities and organs of the body. Epithelium isderived embryologically from ectoderm, endoderm and mesoderm. To beclassified as adenocarcinoma, the cells do not necessarily need to bepart of a gland, as long as they have secretory properties. This form ofcarcinoma can occur in some higher mammals, including humans. Welldifferentiated adenocarcinomas tend to resemble the glandular tissuethat they are derived from, while poorly differentiated may not. Bystaining the cells from a biopsy, a pathologist will determine whetherthe tumor is an adenocarcinoma or some other type of cancer.Adenocarcinomas can arise in many tissues of the body due to theubiquitous nature of glands within the body. While each gland may not besecreting the same substance, as long as there is an exocrine functionto the cell, it is considered glandular and its malignant form istherefore named adenocarcinoma. Malignant adenocarcinomas invade othertissues and often metastasize given enough time to do so. Ovarianadenocarcinoma is the most common type of ovarian carcinoma. It includesthe serous and mucinous adenocarcinomas, the clear cell adenocarcinomaand the endometrioid adenocarcinoma.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor, i.e. a secondary tumor ormetastatic tumor, at the target site depends on angiogenesis. Tumormetastasis often occurs even after the removal of the primary tumorbecause tumor cells or components may remain and develop metastaticpotential. In one embodiment, the term “metastasis” according to theinvention relates to “distant metastasis” which relates to a metastasiswhich is remote from the primary tumor and the regional lymph nodesystem.

The cells of a secondary or metastatic tumor are like those in theoriginal tumor. This means, for example, that, if breast cancermetastasizes to the liver, the secondary tumor is made up of abnormalbreast cells, not of abnormal liver cells. The tumor in the liver isthen called metastatic breast cancer, not liver cancer.

The term “circulating tumor cells” or “CTCs” relates to cells that havedetached from a primary tumor or tumor metastases and circulate in thebloodstream. CTCs may constitute seeds for subsequent growth ofadditional tumors (metastasis) in different tissues. Circulating tumorcells are found in frequencies in the order of 1-10 CTC per mL of wholeblood in patients with metastatic disease. Research methods have beendeveloped to isolate CTC. Several research methods have been describedin the art to isolate CTCs, e.g. techniques which use of the fact thatepithelial cells commonly express the cell adhesion protein EpCAM, whichis absent in normal blood cells. Immunomagnetic bead-based captureinvolves treating blood specimens with antibody to EpCAM that has beenconjugated with magnetic particles, followed by separation of taggedcells in a magnetic field. Isolated cells are then stained with antibodyto another epithelial marker, cytokeratin, as well as a common leukocytemarker CD45, so as to distinguish rare CTCs from contaminating whiteblood cells. This robust and semi-automated approach identifies CTCswith an average yield of approximately 1 CTC/mL and a purity of 0.1%(Allard et al., 2004: Clin Cancer Res 10, 6897-6904). A second methodfor isolating CTCs uses a microfluidic-based CTC capture device whichinvolves flowing whole blood through a chamber embedded with 80,000microposts that have been rendered functional by coating with antibodyto EpCAM. CTCs are then stained with secondary antibodies against eithercytokeratin or tissue specific markers, such as PSA in prostate canceror HER2 in breast cancer and are visualized by automated scanning ofmicroposts in multiple planes along three dimensional coordinates.CTC-chips are able to identifying cytokerating-positive circulatingtumor cells in patients with a median yield of 50 cells/ml and purityranging from 1-80% (Nagrath et al., 2007: Nature 450, 1235-1239).Another possibility for isolating CTCs is using the CellSearch™Circulating Tumor Cell (CTC) Test from Veridex, LLC (Raritan, N.J.)which captures, identifies, and counts CTCs in a tube of blood. TheCellSearch™ system is a U.S. Food and Drug Administration (FDA) approvedmethodology for enumeration of CTC in whole blood which is based on acombination of immunomagnetic labeling and automated digital microscopy.There are other methods for isolating CTCs described in the literatureall of which can be used in conjunction with the present invention.

A relapse or recurrence occurs when a person is affected again by acondition that affected them in the past. For example, if a patient hassuffered from a tumor disease, has received a successful treatment ofsaid disease and again develops said disease said newly developeddisease may be considered as relapse or recurrence. However, accordingto the invention, a relapse or recurrence of a tumor disease may butdoes not necessarily occur at the site of the original tumor disease.Thus, for example, if a patient has suffered from breast tumor and hasreceived a successful treatment a relapse or recurrence may be theoccurrence of a breast tumor or the occurrence of a tumor at a sitedifferent to breast. A relapse or recurrence of a tumor also includessituations wherein a tumor occurs at a site different to the site of theoriginal tumor as well as at the site of the original tumor. Preferably,the original tumor for which the patient has received a treatment is aprimary tumor and the tumor at a site different to the site of theoriginal tumor is a secondary or metastatic tumor.

By “treat” is meant to administer a compound or composition as describedherein to a subject in order to prevent or eliminate a disease,including reducing the size of a tumor or the number of tumors in asubject; arrest or slow a disease in a subject; inhibit or slow thedevelopment of a new disease in a subject; decrease the frequency orseverity of symptoms and/or recurrences in a subject who currently hasor who previously has had a disease; and/or prolong, i.e. increase thelifespan of the subject. In particular, the term “treatment of adisease” includes curing, shortening the duration, ameliorating,preventing, slowing down or inhibiting progression or worsening, orpreventing or delaying the onset of a disease or the symptoms thereof.

By “being at risk” is meant a subject, i.e. a patient, that isidentified as having a higher than normal chance of developing adisease, in particular cancer, compared to the general population. Inaddition, a subject who has had, or who currently has, a disease, inparticular cancer, is a subject who has an increased risk for developinga disease, as such a subject may continue to develop a disease. Subjectswho currently have, or who have had, a cancer also have an increasedrisk for cancer metastases.

The term “immunotherapy” relates to a treatment involving a specificimmune reaction.

In the context of the present invention, terms such as “protect”,“prevent”, “prophylactic”, “preventive”, or “protective” relate to theprevention or treatment or both of the occurrence and/or the propagationof a disease in a subject and, in particular, to minimizing the chancethat a subject will develop a disease or to delaying the development ofa disease. For example, a person at risk for a tumor, as describedabove, would be a candidate for therapy to prevent a tumor.

A prophylactic administration of an immunotherapy, for example, aprophylactic administration of a vaccine of the invention, preferablyprotects the recipient from the development of a disease. A therapeuticadministration of an immunotherapy, for example, a therapeuticadministration of a vaccine of the invention, may lead to the inhibitionof the progress/growth of the disease. This comprises the decelerationof the progress/growth of the disease, in particular a disruption of theprogression of the disease, which preferably leads to elimination of thedisease.

Immunotherapy may be performed using any of a variety of techniques, inwhich agents provided herein function to remove diseased cells from apatient. Such removal may take place as a result of enhancing orinducing an immune response in a patient specific for an antigen or acell expressing an antigen.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against diseased cells with theadministration of immune response-modifying agents (such as polypeptidesand nucleic acids as provided herein).

The agents and compositions provided herein may be used alone or incombination with conventional therapeutic regimens such as surgery,irradiation, chemotherapy and/or bone marrow transplantation(autologous, syngeneic, allogeneic or unrelated).

The term “immunization” or “vaccination” describes the process oftreating a subject with the purpose of inducing an immune response fortherapeutic or prophylactic reasons.

The term “in vivo” relates to the situation in a subject.

The terms “subject”, “individual”, “organism” or “patient” are usedinterchangeably and relate to vertebrates, preferably mammals. Forexample, mammals in the context of the present invention are humans,non-human primates, domesticated animals such as dogs, cats, sheep,cattle, goats, pigs, horses etc., laboratory animals such as mice, rats,rabbits, guinea pigs, etc. as well as animals in captivity such asanimals of zoos. The term “animal” as used herein also includes humans.The term “subject” may also include a patient, i.e., an animal,preferably a human having a disease, preferably a disease as describedherein.

In one embodiment, a method of the invention is performed on a patientwhich is already diagnosed as having cancer.

The term “autologous” is used to describe anything that is derived fromthe same subject. For example, “autologous transplant” refers to atransplant of tissue or organs derived from the same subject. Suchprocedures are advantageous because they overcome the immunologicalbarrier which otherwise results in rejection.

The term “heterologous” is used to describe something consisting ofmultiple different elements. As an example, the transfer of oneindividual's bone marrow into a different individual constitutes aheterologous transplant. A heterologous gene is a gene derived from asource other than the subject.

As part of the composition for an immunization or a vaccination,preferably one or more agents as described herein are administeredtogether with one or more adjuvants for inducing an immune response orfor increasing an immune response. The term “adjuvant” relates tocompounds which prolongs or enhances or accelerates an immune response.The composition of the present invention preferably exerts its effectwithout addition of adjuvants. Still, the composition of the presentapplication may contain any known adjuvant. Adjuvants comprise aheterogeneous group of compounds such as oil emulsions (e.g., Freund'sadjuvants), mineral compounds (such as alum), bacterial products (suchas Bordetella pertussis toxin), liposomes, and immune-stimulatingcomplexes. Examples for adjuvants are monophosphoryl-lipid-A (MPLSmithKline Beecham). Saponins such as QS21 (SmithKline Beecham), DQS21(SmithKline Beecham; WO 96/33739), QS7, QS17, QS18, and QS-L1 (So et al,1997, Mol. Cells 7: 178-186), incomplete Freund's adjuvants, completeFreund's adjuvants, vitamin E, montanid, alum, CpG oligonucleotides(Krieg et al., 1995, Nature 374: 546-549), and various water-in-oilemulsions which are prepared from biologically degradable oils such assqualene and/or tocopherol.

Other substances which stimulate an immune response of the patient mayalso be administered. It is possible, for example, to use cytokines in avaccination, owing to their regulatory properties on lymphocytes. Suchcytokines comprise, for example, interleukin-12 (IL-12) which was shownto increase the protective actions of vaccines (cf. Science268:1432-1434, 1995), GM-CSF and IL-18.

There are a number of compounds which enhance an immune response andwhich therefore may be used in a vaccination. Said compounds compriseco-stimulating molecules provided in the form of proteins or nucleicacids such as B7-1 and B7-2 (CD80 and CD86, respectively).

According to the invention, a sample is preferably a bodily sample. Abodily sample may be a tissue sample, including body fluids, and/or acellular sample. Such bodily samples may be obtained in the conventionalmanner such as by tissue biopsy, including punch biopsy, and by takingblood, bronchial aspirate, sputum, urine, feces or other body fluids.According to the invention, the term “sample” also includes processedsamples such as fractions or isolates of biological samples, e.g.nucleic acid or cell isolates.

The agents such as vaccines and compositions described herein may beadministered via any conventional route, including by injection orinfusion. The administration may be carried out, for example, orally,intravenously, intraperitoneally, intramuscularly, subcutaneously ortransdermally. In one embodiment, administration is carried outintranodally such as by injection into a lymph node. Other forms ofadministration envision the in vitro transfection of antigen presentingcells such as dendritic cells with nucleic acids described hereinfollowed by administration of the antigen presenting cells.

The agents described herein are administered in effective amounts. An“effective amount” refers to the amount which achieves a desiredreaction or a desired effect alone or together with further doses. Inthe case of treatment of a particular disease or of a particularcondition, the desired reaction preferably relates to inhibition of thecourse of the disease. This comprises slowing down the progress of thedisease and, in particular, interrupting or reversing the progress ofthe disease. The desired reaction in a treatment of a disease or of acondition may also be delay of the onset or a prevention of the onset ofsaid disease or said condition.

An effective amount of an agent described herein will depend on thecondition to be treated, the severeness of the disease, the individualparameters of the patient, including age, physiological condition, sizeand weight, the duration of treatment, the type of an accompanyingtherapy (if present), the specific route of administration and similarfactors. Accordingly, the doses administered of the agents describedherein may depend on various of such parameters. In the case that areaction in a patient is insufficient with an initial dose, higher doses(or effectively higher doses achieved by a different, more localizedroute of administration) may be used.

The pharmaceutical compositions described herein are preferably sterileand contain an effective amount of the therapeutically active substanceto generate the desired reaction or the desired effect.

The pharmaceutical compositions described herein are generallyadministered in pharmaceutically compatible amounts and inpharmaceutically compatible preparation. The term “pharmaceuticallycompatible” refers to a nontoxic material which does not interact withthe action of the active component of the pharmaceutical composition.Preparations of this kind may usually contain salts, buffer substances,preservatives, carriers, supplementing immunity-enhancing substancessuch as adjuvants, e.g. CpG oligonucleotides, cytokines, chemokines,saponin, GM-CSF and/or RNA and, where appropriate, other therapeuticallyactive compounds. When used in medicine, the salts should bepharmaceutically compatible. However, salts which are notpharmaceutically compatible may used for preparing pharmaceuticallycompatible salts and are included in the invention. Pharmacologicallyand pharmaceutically compatible salts of this kind comprise in anon-limiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallycompatible salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

A pharmaceutical composition described herein may comprise apharmaceutically compatible carrier. The term “carrier” refers to anorganic or inorganic component, of a natural or synthetic nature, inwhich the active component is combined in order to facilitateapplication. According to the invention, the term “pharmaceuticallycompatible carrier” includes one or more compatible solid or liquidfillers, diluents or encapsulating substances, which are suitable foradministration to a patient. The components of the pharmaceuticalcomposition of the invention are usually such that no interaction occurswhich substantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions described herein may contain suitablebuffer substances such as acetic acid in a salt, citric acid in a salt,boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may, where appropriate, also containsuitable preservatives such as benzalkonium chloride, chlorobutanol,paraben and thimerosal.

The pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. Pharmaceuticalcompositions of the invention may be in the form of capsules, tablets,lozenges, solutions, suspensions, syrups, elixirs or in the form of anemulsion, for example.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

According to the invention, expression, such as expression of a tumorantigen or a set of tumor antigens, may be determined on the mRNA level(transcriptional level) or protein level (translational level), forexample, by measuring the transcribed mRNA (e.g. via northern blot), bymeasuring the produced protein (e.g. via Western Blot), by directly orindirectly staining the protein (e.g. via immunohistochemistry) or bydirectly staining the mRNA (e.g. via in situ hybridization).

For example, according to the invention, determining the expression of atumor antigen on the mRNA level (transcriptional level) may be carriedout by detecting and/or determining the quantity of any of the nucleicacid sequences described herein for the respective tumor antigen. Forexample, determining the expression of CAGE1 may be carried out bydetecting and/or determining the quantity of the nucleic acid sequenceof SEQ ID NO: 41 or 42 or a variant thereof.

For example, according to the invention, determining the expression of atumor antigen on the protein level (translational level) may be carriedout by detecting and/or determining the quantity of any of the aminoacid sequences described herein for the respective tumor antigen. Forexample, determining the expression of CAGE1 may be carried out bydetecting and/or determining the quantity of the amino acid sequence ofSEQ ID NO: 43 or 44 or a variant thereof.

In one preferred embodiment, the expression level is determined using animmunoassay, gel electrophoresis, spectrometry, chromatography, in situhybridization, or a combination thereof.

According to the invention, an immunoassay may be selected from thegroup consisting of western blots, immunohistochemistry,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitation reactions, geldiffusion precipitation reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, immunofluorescence, protein A immunoassays,flow cytometry and FACS analysis.

The present invention further relates to a kit comprising means such asreagents for determining the expression pattern of a set of tumorantigens, preferably in a sample isolated from a cancer patient. Saidkit is useful for conducting the methods of the present invention.

In the context of the present invention, the term “kit of parts (inshort: kit)” is understood to be any combination of at least some of thecomponents identified herein, which are combined, coexisting spatially,to a functional unit, and which can contain further components.

In one embodiment, the kit comprises one or more means that specificallybind to tumor antigens of a set of tumor antigens.

Said means that specifically bind to tumor antigens may be antibodies orfragments thereof, which are capable of specially binding to an epitopeor a suitable structural element of an antigen to be detected.

In other embodiments, said means may be nucleic acid. For nucleic aciddetection, the kits generally comprise (but are not limited to) probesspecific for mRNA encoding tumor antigens. For Quantitative PCR, thekits generally comprise pre-selected primers specific for nucleic acidsequences encoding tumor antigens. The Quantitative PCR kits may alsocomprise enzymes suitable for amplifying nucleic acids (e.g.,polymerases such as Taq), and deoxynucleotides and buffers needed forthe reaction mixture for amplification. The Quantitative PCR kits mayalso comprise probes specific for nucleic acid sequences encoding tumorantigens. In some embodiments, the Quantitative PCR kits also comprisecomponents suitable for reverse-transcribing RNA including enzymes (e.g.reverse transcriptases) and primers for reverse transcription along withdeoxynucleotides and buffers needed for the reverse transcriptionreaction.

In certain embodiments, said means are detectably labeled.

The antibody or antibody fragment may be bound to a solid support, e.g.a plastic surface, to allow binding and detection of a protein. Forexample, a microtiter plate can be used as a plastic surface. Thedetection of the binding can be effected by using a secondary antibodylabeled with a detectable group. The detectable group can be an enzymelike horseradish peroxidase (HRP) or alkaline phosphatase detectable byadding a suitable substrate to produce a colour or a fluorescencesignal.

Said kit may further comprise (i) a container, and/or (ii) a datacarrier. Said container may be filled with one or more of the abovementioned means or reagents. Said data carrier may be a non-electronicaldata carrier, e.g. a graphical data carrier such as an informationleaflet, an information sheet, a bar code or an access code, or anelectronical data carrier such as a floppy disk, a compact disk (CD), adigital versatile disk (DVD), a microchip or another semiconductor-basedelectronical data carrier. The access code may allow the access to adatabase, e.g. an internet database, a centralized, or a decentralizeddatabase. Said data carrier may comprise instructions for the use of thekit in the methods of the invention.

Additionally or alternatively, said kit may comprise materials desirablefrom a commercial and user standpoint including buffer(s), reagent(s)and/or diluent(s) for determining expression of protein or mRNA.

The above mentioned data carrier may comprise a threshold value orreference level of a protein or mRNA. In case that the data carriercomprises an access code which allows the access to a database, saidthreshold value or reference level is deposited in this database.

In addition, the data carrier may comprise information or instructionson how to carry out the methods of the present invention.

The teaching given herein with respect to specific amino acid sequences,e.g. those shown in the sequence listing, is to be construed so as toalso relate to variants of said specific sequences resulting insequences which are functionally equivalent to said specific sequences,e.g. amino acid sequences exhibiting properties identical or similar tothose of the specific amino acid sequences.

The term “variant” according to the invention refers, in particular, tomutants, splice variants, conformations, isoforms, allelic variants,species variants and species homologs, in particular those which arenaturally present. An allelic variant relates to an alteration in thenormal sequence of a gene, the significance of which is often unclear.Complete gene sequencing often identifies numerous allelic variants fora given gene. A species homolog is a nucleic acid or amino acid sequencewith a different species of origin from that of a given nucleic acid oramino acid sequence. The term “variant” shall encompass anyposttranslationally modified variants and conformation variants.

For the purposes of the present invention, “variants” of an amino acidsequence comprise amino acid insertion variants, amino acid additionvariants, amino acid deletion variants and/or amino acid substitutionvariants.

Preferably the degree of similarity, preferably identity between a givenamino acid sequence and an amino acid sequence which is a variant ofsaid given amino acid sequence will be at least about 60%, 65%, 70%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity isgiven preferably for an amino acid region which is at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90% or about 100% of the entire length of the referenceamino acid sequence. For example, if the reference amino acid sequenceconsists of 200 amino acids, the degree of similarity or identity isgiven preferably for at least about 20, at least about 40, at leastabout 60, at least about 80, at least about 100, at least about 120, atleast about 140, at least about 160, at least about 180, or about 200amino acids, preferably continuous amino acids. In preferredembodiments, the degree of similarity or identity is given for theentire length of the reference amino acid sequence. The alignment fordetermining sequence similarity, preferably sequence identity can bedone with art known tools, preferably using the best sequence alignment,for example, using Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two amino acid sequencesindicates the percentage of amino acids that are identical between thesequences.

The term “percentage identity” is intended to denote a percentage ofamino acid residues which are identical between the two sequences to becompared, obtained after the best alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly and over their entire length. Sequence comparisonsbetween two amino acid sequences are conventionally carried out bycomparing these sequences after having aligned them optimally, saidcomparison being carried out by segment or by “window of comparison” inorder to identify and compare local regions of sequence similarity. Theoptimal alignment of the sequences for comparison may be produced,besides manually, by means of the local homology algorithm of Smith andWaterman, 1981, Ads App. Math. 2, 482, by means of the local homologyalgorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by meansof the similarity search method of Pearson and Lipman, 1988, Proc. NatlAcad. Sci. USA 85, 2444, or by means of computer programs which usethese algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA inWisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

With respect to nucleic acid molecules, the term “variant” includesdegenerate nucleic acid sequences, wherein a degenerate nucleic acidaccording to the invention is a nucleic acid that differs from areference nucleic acid in codon sequence due to the degeneracy of thegenetic code.

Furthermore, a “variant” of a given nucleic acid sequence according tothe invention includes nucleic acid sequences comprising single ormultiple such as at least 2, at least 4, or at least 6 and preferably upto 3, up to 4, up to 5, up to 6, up to 10, up to 15, or up to 20nucleotide substitutions, deletions and/or additions.

Preferably the degree of identity between a given nucleic acid sequenceand a nucleic acid sequence which is a variant of said given nucleicacid sequence will be at least 70%, preferably at least 75%, preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90% or most preferably at least 95%, 96%, 97%, 98% or 99%. Thedegree of identity is preferably given for a region of at least about30, at least about 50, at least about 70, at least about 90, at leastabout 100, at least about 150, at least about 200, at least about 250,at least about 300, or at least about 400 nucleotides. In preferredembodiments, the degree of identity is given for the entire length ofthe reference nucleic acid sequence.

“Sequence identity” between two nucleic acid sequences indicates thepercentage of nucleotides that are identical between the sequences.

The term “percentage identity” is intended to denote a percentage ofnucleotides which are identical between the two sequences to becompared, obtained after the best alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly and over their entire length. Sequence comparisonsbetween two nucleotide sequences are conventionally carried out bycomparing these sequences after having aligned them optimally, saidcomparison being carried out by segment or by “window of comparison” inorder to identify and compare local regions of sequence similarity. Theoptimal alignment of the sequences for comparison may be produced,besides manually, by means of the local homology algorithm of Smith andWaterman, 1981, Ads App. Math. 2, 482, by means of the local homologyalgorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by meansof the similarity search method of Pearson and Lipman, 1988, Proc. NatlAcad. Sci. USA 85, 2444, or by means of computer programs which usethese algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA inWisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting. Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

FIGURES

FIG. 1: Analysis of tumor antigen expression in breast cancer

FIG. 2: Analysis of tumor antigen expression in triple-negative breastcancer (TNBC)

A combination of only three tumor antigens, CXorf61, CAGE1 and PRAME, issufficient to represent 95% of the analysed samples.

FIG. 3: Box-Whiskers-Plot showing the distribution of transcripts inbreast cancer samples

Distribution of transcripts for CXORF61 (FIG. 3A), PRAME (FIG. 3B), andCAGE1 (FIG. 3C) is shown irrespectively of the subtype(allbreast:tumor), in the breast cancer samples without the TNBC subtype(breast:tumor) and the TNBC subtype.

FIG. 4: Expression of CXORF61 on the protein level

FIG. 5: Tumor specificity of CXORF61, CAGE1 and PRAME

FIG. 6: Identification of T cell epitopes for CXORF61

FIG. 7: Testing of T cell receptors for specificity with T cell epitopesfor CXORF61

FIG. 8: CXorf61 transcript sequence predicted from NGS analysis

The known CXorf61 sequence (NM_001017978.3) is indicated. The newpredicted sequence is in bold.

FIG. 9: The existence of the CXorf61-iso1 transcript can be confirmed byPCR

RT-PCR analysis was performed with the indicated primers (for primersequences see Table 1) using the following cDNAs: 1 Normal testis, 2MDA-MB468, 3 MDA-MB231.

FIG. 10: CXorf6-iso1 is not expressed in normal tissues

The indicated normal tissues (n=65) were analysed by qRTPCR with primersspecific for CXorf61 or CXorf61-iso1. Relative expression was calculatedwith the ΔΔCt method using HPRT as housekeeping gene.

FIG. 11: CXorf61-iso1 is expressed in triple negative breast cancertissues

29 triple negative breast cancer tissues were analysed by qRT-PCR withprimers specific for CXorf61-iso1. Relative expression was calculatedwith the ΔΔCt method using HPRT as housekeeping gene.

FIG. 12: Positions of the new identified CAGE1 exons

As reference the CAGE1 structure as described in UCSC was used. Theposition of the primers used for expression analysis is shown, SEQ IDNO: 45 [NEW EXON 1]: light grey, SEQ ID NO: 46 [NEW EXON 2], dark grey

FIG. 13: Expression of new CAGE1 isoforms in normal tissues

Expression of CAGE1-Tron1 (black columns) and CAGE1-Tron2 (grey columns)was analysed by qRT-PCR with primers 5527+4783 and primers 4782+5540respectively in the indicated normal tissues. A positive tumor tissuewas used as control. Relative expression was calculated with the ΔΔCtmethod using HPRT as housekeeping gene.

FIG. 14: Expression of new CAGE1 isoforms in cancer tissues

34 Tissues from Triple negatives breast cancer patients were analysed byqRT-PCR with primers specific for CAGE1-Tron1 (primers 5527+4783, darkcolumns) and CAGE1-Tron2 (primers 4782+5540, grey columns) respectively.Relative expression was calculated with the ΔΔCt method using HPRT ashousekeeping gene.

EXAMPLES

The techniques and methods used herein are described herein or carriedout in a manner known per se and as described, for example, in Sambrooket al., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Allmethods including the use of kits and reagents are carried out accordingto the manufacturers' information unless specifically indicated.

Example 1: Establishing a Set of Tumor Antigens Useful in a LargeFraction of Cancer Patients

It was assessed whether it is possible to establish a set of tumorantigens which is shared at least partially by a large fraction of tumorpatients and on the basis of which a set of vaccine products applicableto a broad spectrum of cancer patients can be provided.

To this end, RNA was extracted from normal tissues or breast cancersamples using the RNeasy Lipid Tissue Mini Kit (Qiagen). cDNA synthesiswas performed using the SuperScript II Reverse Transcriptase Kit(Invitrogen) and oligo-dT. Expression was analysed using the BioMark™ HDSystem system (Fluidigm) and the relative expression was calculatedusing HPRT as house keeping gene.

In this manner, the relative expression of several genes could bedetected. The % of positive samples, using a threshold of relativeexpression of 30000, changed depending on whether all breast cancersamples, irrespectively of the subtype (n=35), were analysed (FIG. 1) oronly the TNBC (n=61) subtype was analysed (FIG. 2). Particularly thethree transcripts PRAME, CXORF61 and CAGE1, are sufficient to representabout 95% of the analysed patient samples in the TNBC group and 35% inthe all breast cancer group. The distribution of the three transcriptsin the breast cancer samples, irrespectively of the subtype(allbreast:tumor), in the breast cancer samples without the TNBC subtype(breast:tumor) and the TNBC subtype is shown as Box-Whiskers-Plot inFIG. 3A (CXORF61), FIG. 3B (PRAME), FIG. 3C (CAGE1).

FIG. 4 shows expression of CXORF61 also on the protein level. For theanalysis, total protein extracts were generated from 8 human TNBCsamples and normalized to β-actin (FIG. 4, lowest panel). CXORF61expression was analyzed by immunoblotting with a specific antibody (FIG.4, upper panel). A negative cell lines (Hek-mock) and a positive cellline, Hek transfected with a plasmid coding for CXORF61 (Hek-CXORf61)were used as control. CXORF6 is detectable in 5 out of 8 testedtriple-negative breast cancer samples (FIG. 4, lanes 3-5, 7-8).

Example 2: Analysis of Tumor Specificity of CXORF61, CAGE1 and PRAME

The tumor specificity of the three transcripts was analysed by qRT-PCRin a large set of normal tissues (n=65). As shown in FIG. 5, highexpression (>30.000) of CXORF61 was detectable only in testis, highexpression of PRAME was detectable in testis, endometrium andepididymis. CAGE1 shows only weak expression in testis. In contrast,Erbb2, a generally accepted target for vaccination and otherimmunotherapeutic approaches, shows a high expression in several normaltissues.

Example 3: T Cell Epitopes of CXORF61, CAGE1 and PRAME

For CXORF61, TCR epitopes were identified by ex-vivo reactivity,analyzed by IFNγ-ELISPOT assay, of spleen cells from CXORF6 immunizedHLA-A*02-transgenic mice against CXORF61-derived peptides. To this end,HLA-A*02 binding peptides derived from CXORF61 were predicted applyingthe SYFPEITHY algorithm (FIG. 6A). Spleen cells were analyzed forreactivity against CXORF61 peptide pool or predicted HLA-A*02-bindingCXORF61-derived peptides A2-1-6 (FIG. 6B). Positive control: PMA-treatedspleen cells; negative control: an irrelevant peptide pool (HIV-gag),irrelevant nonamer peptide (PLAC1-31-39). Two A2-restricted epitopeswere identified (peptide A2-2 and A2-4).

After isolation of TCRs from CD8+ T cells of CXORF61-immunized mice,CD8+ T cells of a HLA-A*02-positive healthy donor were transfected withTCR-α/β chain RNAs and tested by IFNγ-ELISPOT assay for recognition ofK562-A2 cells transfected with CXORF61 RNA or pulsed with CXORF61overlapping 15mer peptides (=CXORF61 pool) or HLA-A*02 binding peptidesCXORF61-A2-2/4 (FIG. 7). Negative controls: irrelevant peptide pool(HIV-gag), irrelevant 9mer peptide (Plac1-31-39); Positive control: SEB.Three TCRs were specific for peptide A-2 and one TCR for peptide A2-4.

For PRAME a large number of T cell epitopes has been reported to elicitantitumoral cytotoxic T cell responses. In a publication by Kessler etal. (2001, J. Exp. Med. 193(1):73-88) four HLA-A*0201-presentedcytotoxic T lymphocyte (CTL) PRAME epitopes (VLDGLDVLL, SLYSFPEPEA,ALYVDSLFFL, and SLLQHLIGL) are reported. The publication demonstratedthe lysis of mammary carcinoma cell lines for these epitopes. FurtherPRAME epitopes eliciting a immune response by cytotoxic T lymphocyteshave been identified by Kessler et al. (2003, Hum Immunol. 64(2):245-55)(LPRELFPPL, LPRRLFPPLF, FPPLFMAAF, IPVEVLVDLF, LPTLAKFSPY, CPHCGDRTFY,EPILCPCFM, HLA-B35), Kawahara et al. (2006, Exp Hematol.34(11):1496-504) (GQHLHLETF, HLA-B*62), and Quintarelli et al. (2011,Blood 117(12):3353-62) (NLTHVLYPV, HLA-A*02).

For CAGE1 a publication about serological analysis of cDNA expressionlibraries (SEREX) showed that cancer patients specifically developedauto-antibodies against this target (Park et al., 2003, Biochim BiophysActa. 1625(2):173-82). Induction of cytolytic T lymphocyte (CTL)reactions has been reported for several cancer/testis antigens.

Example 4: Identification of a CXorf61 Isoform Strictly Expressed inTumor Tissues

A new CXorf61 transcript was identified by Next Generation Sequencing,which is expressed in tumor cell lines but not in normal testis. Thissequence has a longer 3′ UTR and does not change the CXorf61 OpenReading Frame (ORF) (FIG. 8).

To confirm the existence of a longer CXorf61 transcript, the predictedsequence was amplified using primers binding at the 5′ of the knownsequence and in the new predicted 3′ (primers 3873+3875) (FIG. 9). Ascontrol, primers binding in NM_001017978.3 were used (primers3873+3874). While the NM_001017978.3 sequence can be detected in normaltestis and breast tumor cell lines MDA-MB468 and MDA-MB231, the newsequence can be detected only in the breast tumor cell lines. The PCRproduct was sent to sequencing, confirming the existence of thepredicted sequence.

Relative expression of CXorf61-iso1 was analyzed by qRT-PCR using theFluidigm system (primers 2898+3876). While expression of CXorf61(primers 2898+2899) was detectable very strongly in normal testis andweakly in epididymis and placenta, only irrelevant expression in normaltestis was found for CXorf61-iso1 (FIG. 10).

Relative expression of CXorf61-iso1 was analyzed by qRT-PCR using theFluidigm system in triple negative breast cancer patient samples withprimers 2898+3876 (FIG. 11). Using a threshold of 10000, 59% of thesamples were positive for CXorf61 iso1.

TABLE 1 Sequence of the primers used Primer Sequence (5′-3′) 3873CCGTTTCCCAAATCCAGGC 3874 ATCTACTCAAAGTGTCTTTAATGATTTCC 2898GTGTGCCTTGATTGTCTTCTGG 3875 CTTTCTCTATTGTGCTTCCATTCC 3876GTATCTGGATTTTTTGTATGTGACTTGGAAT 2899 CCTGGCTATTGAGTGTGGG

Example 5: Identification of CAGE1 Isoforms Strictly Expressed in TumorTissues

By using Next Generation sequencing an alternative start codon in theCAGE1 sequence was identified. The existence of the new exon wasconfirmed by RT-PCR and sequencing. RT-PCR analysis was performed withprimers 5274 and 5527 (for primers sequence see Table 2, for primersposition see FIG. 12) using the cDNA of a Triple negative breast cancerpatient. The amplified products were extracted from the gel and weresequenced.

TABLE 2 Sequence of the primers used for expression analysis Primernumber Sequence (5′-3′) 5274 GACTCTTCCTGGAGTGGTTGA 5540GAACCCCGGAAGTGGAGGTT 4783 GGTCATGGACTTCGGATGATT 4782AGGATTTAATTAGAAAGCCCAGAGA 5527 CTCTACCCCTGTATTTCGCTTG

The position of the primers used for expression analysis is shown inFIG. 12. The light grey bar indicates the exon at 5′ and the dark greybar the exon at 3′ of the same figure. The new isoforms are shorter asthe known CAGE1 isoforms. The new identified CAGE1 isoforms are SEQ IDNO: 41 [CAGE1-TRON1] and 42 [CAGE1-TRON2] encoding for the predictedORFs of SEQ ID NO: 43 [CAGE1-TRON1-ORF] and 44 [CAGE1-TRON2-ORF],respectively. CAGE1-TRON1 comprises the new exon of SEQ ID NO: 45 andCAGE1-TRON2 comprises the new exons SEQ ID NO: 45 and 46. As referencethe CAGE1 structure as described in UCSC was used:

TABLE 3 CAGE1 reference sequences CAGE1 (uc021y1c.1) at chr6:7326887-7389942-Homo sapiens cancer antigen 1 (CAGE1), transcriptvariant 3, mRNA. CAGE1 (uc003mxl.2) at chr6: 7326887-7389942-Homosapiens cancer antigen 1 (CAGE1), transcript variant 1, mRNA. CAGE1(uc003mxk.2) at chr6: 7329330-7389942-Homo sapiens cancer antigen 1(CAGE1), transcript-variant 2, mRNA, CAGE1 (uc003mxj.3) at chr6:7326887-7389942-Homo sapiens cancer antigen 1 (CAGE1), transcriptvariant 1, mRNA. CAGE1 (uc003mxh.3) at chr6: 7326887-7374364-Homosapiens cancer antigen 1 (CAGE1), transcript variant 1, mRNA.

CAGE1-Tron1 and CAGE1-Tron2 have a restricted expression in normaltissues. Relative expression of CAGE1-Tron1 and CAGE1-Tron2 was analysedin 60 normal tissues, including several brain areas by qRT-PCR using theFluidigm technology. Expression of CAGE1-Tron1 was detected in placentaand at less extent in testis. No expression of CAGE1-Tron2 was detectedin any normal tissues (FIG. 13).

CAGE1-Tron1 and CAGE1-Tron2 are expressed in Triple negative breastcancer tissues. Relative expression of CAGE1-Tron1 and CAGE1-Tron2 wasanalyzed by qRT-PCR in 34 triple negative breast cancer patient samples.Using a cut off of 100, 73% of the tumors are positive for CAGE1-Tron1and 32% for CAGE1-Tron2 (FIG. 14).

The invention claimed is:
 1. A method for treating triple negativebreast cancer in a patient comprising a) detecting the expressionpattern of a set of tumor antigens in a sample from the patient, whereinthe set of tumor antigens comprises CXorf61, CAGE1, and PRAME; b)diagnosing the patient as needing a cancer therapy regimen when CXorf61,CAGE1, and PRAME are expressed; and c) treating the patient with animmunotherapeutic that targets the set of tumor antigens expressed inthe patient.
 2. The method of claim 1 wherein the sample comprisescancer cells.
 3. The method of claim 1 wherein detecting the expressionpattern comprises a quantitative and/or qualitative determination of theexpression of the tumor antigens.
 4. The method of claim 1 whereindetecting the expression pattern comprises determining the expression ofRNA and/or protein of the tumor antigens.
 5. A method for treatingtriple negative breast cancer in a patient comprising administering tosaid patient an immunotherapeutic targeting each tumor antigen of a setof tumor antigens, wherein the set of tumor antigens comprises CXorf6,CAGE1, and PRAME.
 6. The method of claim 5 which comprises inducing acellular immune response in the patient against each tumor antigen ofthe set of tumor antigens.
 7. The method of claim 6 wherein the cellularimmune response is induced by administering a vaccine providing one ormore T cell epitopes of each tumor antigen of the set of tumor antigensto the patient.